Isolated mammalian membrane protein genes; related reagents

ABSTRACT

Nucleic acids encoding various lymphocyte cell proteins from mammalian, including primate, reagents related thereto, including specific antibodies, and purified proteins are described. Methods of using said reagents and related diagnostic kits are also provided.

This application is a divisional application under 37 C.F.R. §1.53(b) ofU.S. application Ser. No. 09/111,470, filed Jul. 8, 1998, now U.S. Pat.No. 6,277,959, which claims priority from Provisional U.S. PatentApplication U.S. Ser. No. 60/053,080, filed Jul. 9, 1997.

FIELD OF THE INVENTION

The present invention contemplates compositions related to genes foundin lymphocytes, e.g., cells which function in the immune system. Thesegenes are useful markers, and may function in controlling development,differentiation, and/or physiology of the mammalian immune system. Inparticular, the application provides nucleic acids, proteins,antibodies, and methods of using them.

BACKGROUND OF THE INVENTION

The circulating component of the mammalian circulatory system comprisesvarious cell types, including red and white blood cells of the erythroidand myeloid cell lineages. See, e.g., Rapaport (1987) Introduction toHematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood:Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul(ed.) (1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.

Dendritic cells (DC) are antigen-processing or presenting cells, and arefound in all tissues of the body. See Steinman (1991) Annual Review ofImmunology 9:271-296; and Banchereau and Schmitt (eds. 1994) DendriticCells in Fundamental and Clinical Immunology Plenum Press, NY. These DCcan be classified into various categories, including: interstitialdendritic cells of the heart, kidney, gut, and lung; Langerhans cells inthe skin and mucous membranes; interdigitating dendritic cells in thethymic medula and secondary lymphoid tissue; and blood and lymphdendritic cells. Although dendritic cells in each of these compartmentsare CD45+ leukocytes that apparently arise from bone marrow, they mayexhibit differences that relate to maturation state andmicroenvironment.

These dendritic cells efficiently process and present antigens to, e.g.,T cells. They stimulate responses from naive and memory T cells in theparacortical area of secondary lymphoid organs. There is some evidencefor a role in induction of tolerance.

The primary and secondary B-cell follicles contain follicular dendriticcells that trap and retain intact antigen as immune complexes for longperiods of time. These dendritic cells present native antigen to B cellsand are likely to be involved in the affinity maturation of antibodies,the generation of immune memory, and the maintenance of humoral immuneresponses.

Monocytes are phagocytic cells that belong to the mononuclear phagocytesystem and reside in the circulation. See Roitt (ed) Encyclopedia ofImmunology Academic Press, San Diego. These cells originate in the bonemarrow and remain only a short time in the marrow compartment once theydifferentiate. They then enter the circulation and can remain there fora relatively long period of time, e.g., a few days. The monocytes canenter the tissues and body cavities by the process designateddiapedesis, where they differentiate into macrophages and possibly intodendritic cells. In an inflammatory response, the number of monocytes inthe circulation may double, and many of the increased number ofmonocytes diapedese to the site of inflammation.

Antigen presentation refers to the cellular events in which aproteinaceous antigen is taken up, processed by antigen presenting cells(APC), and then recognized to initiate an immune response. The mostactive antigen presenting cells have been characterized as themacrophages, which are direct developmental products from monocytes;dendritic cells; and certain B cells.

Macrophages are found in most tissues and are highly active ininternalization of a wide variety of protein antigens andmicroorganisms. They have a highly developed endocytic activity, andsecrete many products important in the initiation of an immune response.For this reason, it is believed that many genes expressed by monocytesor induced by monocyte activation are likely to be important in antigenuptake, processing, presentation, or regulation of the resulting immuneresponse.

However, dendritic cells and monocytes are poorly characterized, both interms of proteins they express, and many of their functions andmechanisms of action, including their activated states. In particular,the processes and mechanisms related to the initiation of an immuneresponse, including antigen pocessing and presentation, remain unclear.The absence of knowledge about the structural, biological, andphysiological properties of these cells limits their understanding.Thus, medical conditions where regulation, development, or physiology ofantigen presenting cells is unusual remain unmanageable.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of variousmammalian Dendritic Cell Membrane Protein (DCMP) genes, exemplified bythe specific DCMP1 and DCMP2 embodiments. Distribution data indicates abroader cellular distribution, and structural data suggests somefunction. The DCMP1 exhibits similarity to a class of lectins andasialoglycoprotein receptors. The DCMP2 embodiments described exhibitsignificant sequence similarity to a macrophage cell asialoglycoproteinreceptor. The invention embraces agonists and antagonists of the geneproducts, e.g., mutations (muteins) of the natural sequences, fusionproteins, chemical mimetics, antibodies, and other structural orfunctional analogs. It is also directed to isolated genes encodingproteins of the invention. Various uses of these different protein ornucleic acid composition are also provided.

In particular embodiments, the invention provides a binding compoundcomprising an antibody binding site which specifically binds to a DCMP1protein; or a polypeptide selected from: Gly Val Ser Glu Leu Gln Glu HisThr Thr Gln Lys Ala His Leu Gly His Cys Pro His Cys Pro Ser Val Cys ValPro (residues 118-144 of SEQ ID NO: 4); Gln Val Ala Thr Leu Asn Asn AsnAla Ser Thr Glu Gly Thr Cys Cys (residues 166-181 of SEQ ID NO: 4); orTrp Lys Pro Gly Gln Pro Asp Asn Trp Gln Gly His Gly Leu Gly (residues263-277 of SEQ ID NO: 4). In preferred embodiments, in the bindingcompound, the antibody binding site is: specifically immunoreactive witha protein of SEQ ID NO: 2 or 8; specifically immunoreactive with aprotein of residues 118 to 144 of SEQ ID NO: 4; raised against apurified or recombinantly produced human DCMP1 protein; raised against apurified or recombinantly produced human protein comprising sequence ofresidues 118 to 144 of SEQ ID NO: 4; in a monoclonal antibody, Fab, orF(ab)2; or the binding compound is: detectably labeled; sterile; or in abuffered composition.

The invention embraces methods using those binding compounds, comprisingcontacting the binding compound with a biological sample comprising anantigen to form a binding compound:antigen complex. In certainembodiments, the biological sample is human, and the binding compound isan antibody. The invention also provides a detection kit comprising suchbinding compound and: instructional material for the use of such bindingcompound for the detection; or a compartment providing segregation ofthe binding compound.

The invention also provides a substantially pure or isolatedpolypeptide, which specifically binds to such binding compounds. Invarious embodiments, the polypeptide: comprises at least a fragment ofat least 14 amino acid residues from a primate DCMP1 protein; comprisesat least 14 amino acids of residues 118 to 144 of SEQ ID NO: 4; is asoluble polypeptide; is detectably labeled; is in a sterile composition;is in a buffered composition; binds to an sialic acid residue; isrecombinantly produced, or has a naturally occurring polypeptidesequence.

Nucleic acid embodiments are provided, including a nucleic acid encodinga polypeptide above, when purified. Often, the nucleic acid: comprisesat least 30 nucleotides of the coding portion of SEQ ID NO: 1 or 7;comprises at least 30 nucleotides from nucleotides 608-688 of SEQ ID NO:3; or comprises at least 30 nucleotides from nucleotides 752-799 of SEQID NO: 3, or it may comprise an insert which selectively hybridizes to anucleic acid encoding a polypeptide defined above. The invention alsoprovides a cell transfected with such a nucleic acid, e.g., whichconsists of the protein encoding portions of SEQ ID NO: 1, 7, or theappropriate portions of SEQ ID NO: 3.

The invention provides methods using at least one strand of thosenucleic acids to form a duplex nucleic acid, comprising a step ofcontacting such strand to a sample to a complementary strand capable ofspecifically hybridizing. In preferred embodiments, the method allowsdetection of the duplex; or allows histological localization of theduplex.

Alternatively, the invention provides methods of using a describedbinding composition, comprising a step of contacting the bindingcomposition with a sample to form a binding composition:antigen complex.In preferred embodiments, the sample is a biological sample, including abody fluid; the antigen is on a cell; or the antigen is furtherpurified.

The invention further embraces methods using those polypeptides,comprising contacting the polypeptide with a sample to form a bindingcomposition:polypeptide complex. In preferred embodiments, thepolypeptide is further purified.

Another method provided is to modulating dendritic cell physiology orfunction comprising a step of contacting the cell with: a bindingcomposition as decribed; a DCMP1 protein as described; or a polypeptideas decribed. The function may also result in initiation or progressionof an immune response. Typically, the contacting is in combination withan antigen, including a cell surface, MHC Class I, or MHC Class IIantigen.

DETAILED DESCRIPTION

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

I. General

The present invention provides DNA sequences encoding mammalian proteinsexpressed on dendritic cells (DC). For a review of dendritic cells, seeSteinman (1991) Annual Review of Immunology 9:271-296; and Banchereauand Schmitt (eds. 1994) Dendritic Cells in Fundamental and ClinicalImmunology Plenum Press, NY. These proteins are designated dendriticcell proteins because they are found on these cells and appear toexhibit some specificity in their expression.

Specific primate, e.g., human, embodiments of these proteins areprovided below. Rodent, e.g., mouse, counterparts also exist. Thedescriptions below are directed, for exemplary purposes, to the human DCgenes, but are likewise applicable to structually, e.g., sequence,related embodiments from other sources or mammalian species, includingpolymorphic or individual variants. These will include, e.g., proteinswhich exhibit a relatively few changes in sequence, e.g., less thanabout 5%, and number, e.g., less than 20 residue substitutions,typically less than 15, preferably less than 10, and more preferablyless than 5 substitutions, including 4, 3, 2, or 1. These will alsoinclude versions which are truncated from full length, as described, andfusion proteins containing substantial segments of these sequences.

II. Definitions

The term “binding composition” refers to molecules that bind withspecificity to a these DC proteins, e.g., in an antibody-antigeninteraction. Other compounds, e.g., proteins, can also specificallyassociate with the respective protein. Typically, the specificassociation will be in a natural physiologically relevantprotein-protein interaction, either covalent or non-covalent, and mayinclude members of a multiprotein complex, including carrier compoundsor dimerization partners. The molecule may be a polymer, or chemicalreagent. A functional analog may be a protein with structuralmodifications, or may be a wholly unrelated molecule, e.g., which has amolecular shape which interacts with the appropriate interactingdeterminants. The variants may serve as agonists or antagonists of theprotein, see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's:The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press,Tarrytown, N.Y.

The term “binding agent:DC protein complex”, as used herein, refers to acomplex of a binding agent and DC protein. Specific binding of thebinding agent means that the binding agent has a specific binding sitethat recognizes a site on the respective DC protein. For example,antibodies raised to the DC protein and recognizing an epitope on the DCprotein are capable of forming an antibody:DC protein complex byspecific binding. Typically, the formation of a binding agent:DC proteincomplex allows the measurement of that DC protein in a mixture of otherproteins and biologics. The term “antibody:DC protein complex” refers toa binding agent:DC protein complex in which the binding agent is anantibody. The antibody may be monoclonal, polyclonal or even an antigenbinding fragment of an antibody, e.g., including Fv, Fab, or Fab2fragments.

“Homologous” nucleic acid sequences, when compared, exhibit significantsimilarity. The standards for homology in nucleic acids are eithermeasures for homology generally used in the art by sequence comparisonand/or phylogenetic relationship, or based upon hybridizationconditions. Both algorithms for sequence comparison and hybridizationconditions are described in greater detail below.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany it, e.g., proteins and flanking genomicsequences from the originating species. The term embraces a nucleic acidsequence which has been removed from its naturally occurringenvironment, and includes recombinant or cloned DNA isolates andchemically synthesized analogs or analogs biologically synthesized byheterologous systems. A substantially pure molecule includes isolatedforms of the molecule. An isolated nucleic acid will generally be ahomogeneous composition of molecules, but will, in some embodiments,contain minor heterogeneity. This heterogeneity is typically found atthe polymer ends or portions not critical to a desired biologicalfunction or activity.

As used herein, the term “DCMP1 protein” shall encompass, when used in aprotein context, a protein having amino acid sequences as shown in SEQID NO: 2 or 8, or a significant fragment of such a protein. It refers toa polypeptide which interacts with the respective DCMP1 protein specificbinding components. These binding components, e.g., antibodies,typically bind to the DCMP1 protein with high affinity, e.g., at leastabout 100 nM, usually better than about 30 nM, preferably better thanabout 10 nM, and more preferably at better than about 3 nM.

The term “DCMP2 forms” refers to the sequences provided in SEQ ID NO: 4and 10. The nucleotide and corresponding amino acid sequence of primate,e.g., human, protein related to lectin/asialoglycoprotein familymembers, designated DCMP2, isolated from a dendritic cell library areprovided in SEQ ID NO: 3 and 4. The long form is as shown, while theshort form lacks the sequence corresponding to residues 118-144. Theshort form may also differ at nucleotide 1064. This is related to amonocyte form of an ASGPR, differing by an insertion between residues173 and 174, and at residue 270, see Table 1, and insert of sequenceencoding GEE between nucleotides 775-776. Another variant form isdescribed in SEQ ID NO: 9 and 10.

The term “polypeptide” or “protein” as used herein includes asignificant fragment or segment of said protein, and encompasses astretch of amino acid residues of at least about 8 amino acids,generally at least 10 amino acids, more generally at least 12 aminoacids, often at least 14 amino acids, more often at least 16 aminoacids, typically at least 18 amino acids, more typically at least 20amino acids, usually at least 22 amino acids, more usually at least 24amino acids, preferably at least 26 amino acids, more preferably atleast 28 amino acids, and, in particularly preferred embodiments, atleast about 30 or more amino acids, e.g., 35, 40, 45, 50, 60, 70, etc.

Preferred embodiments exhibit a plurality of distinct, e.g.,nonoverlapping, segments of the specified length. Typically, theplurality will be at least two, more usually at least three, andpreferably 5, 7, or even more. While the length minima are provided,longer lengths, of various sizes, may be appropriate, e.g., one oflength 7, and two of length 12. Segments may refer to either peptides oroligonucleotides.

A “recombinant” nucleic acid is typically defined by its structure. Itcan be a nucleic acid made by generating a sequence comprising fusion oftwo fragments which are not naturally contiguous to each other, but ismeant to exclude products of nature, e.g., naturally occurring mutantforms.

Certain forms are defined by a method of production. In reference tosuch, e.g., a product made by a process, the process is use ofrecombinant nucleic acid techniques, e.g., involving human interventionin the nucleotide sequence, typically selection or production.

Thus, the invention encompasses, for example, nucleic acids comprisingsequence derived using a synthetic oligonucleotide process, and productsmade by transforming cells with a non-naturally occurring vector whichencodes these proteins. Such is often done to replace a codon with aredundant codon encoding the same or a conservative amino acid, whiletypically introducing or removing a sequence recognition site, e.g., fora restriction enzyme. Alternatively, it is performed to join togethernucleic acid segments of desired functions to generate a single geneticentity comprising a desired combination of functions not found in thecommonly available natural forms. Restriction enzyme recognition sitesare often the target of such artificial manipulations, but other sitespecific targets, e.g., promoters, DNA replication sites, regulationsequences, control sequences, or other useful features, e.g., primersegments, may be incorporated by design. A similar concept is intendedfor a recombinant, e.g., fusion, polypeptide. Specifically included aresynthetic nucleic acids which, by genetic code redundancy, encodepolypeptides similar to fragments of these antigens, and fusions ofsequences from various different species variants.

“Solubility” is reflected by sedimentation measured in Svedberg units,which are a measure of the sedimentation velocity of a molecule underparticular conditions. The determination of the sedimentation velocitywas classically performed in an analytical ultracentrifuge, but istypically now performed in a standard ultracentrifuge. See, Freifelder(1982) Physical Biochemistry (2d ed.) W.H. Freeman & Co., San Francisco,Calif.; and Cantor and Schimmel (1980) Biophysical Chemistry parts 1-3,W.H. Freeman & Co., San Francisco, Calif. As a crude determination, asample containing a putatively soluble polypeptide is spun in a standardfull sized ultracentrifuge at about 50K rpm for about 10 minutes, andsoluble molecules will remain in the supernatant. A soluble particle orpolypeptide will typically be less than about 30S, more typically lessthan about 15S, usually less than about 10S, more usually less thanabout 6S, and, in particular embodiments, preferably less than about 4S,and more preferably less than about 3S. Solubility of a polypeptide orfragment depends upon the environment and the polypeptide. Manyparameters affect polypeptide solubility, including temperature,electrolyte environment, size and molecular characteristics of thepolypeptide, and nature of the solvent. Typically, the temperature atwhich the polypeptide is used ranges from about 4° C. to about 65° C.Usually the temperature at use is greater than about 18° C. and moreusually greater than about 22° C. For diagnostic purposes, thetemperature will usually be about room temperature or warmer, but lessthan the denaturation temperature of components in the assay. Fortherapeutic purposes, the temperature will usually be body temperature,typically about 37° C. for humans, though under certain situations thetemperature may be raised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable physiologically active state, and usually not in adenatured state. The polypeptide may be associated with otherpolypeptides in a quaternary structure, e.g., to confer solubility, orassociated with lipids or detergents in a manner which approximatesnatural lipid bilayer interactions.

The solvent will usually be a biologically compatible buffer, of a typeused for preservation of biological activities, and will usuallyapproximate a physiological solvent. Usually the solvent will have aneutral pH, typically between about 5 and 10, and preferably about 7.5.On some occasions, a detergent will be added, typically a mildnon-denaturing one, e.g., e.g., CHS (cholesteryl hemisuccinate) or CHAPS(3-([3-cholamidopropyl]dimethyl-ammonio)-1-propane sulfonate), or in alow enough detergent concentration as to avoid significant disruption ofstructural or physiological properties of the protein.

“Substantially pure” typically means that the protein is isolated fromother contaminating proteins, nucleic acids, or other biologicalsderived from the original source organism. Purity, or “isolation”, maybe assayed by standard methods, typically by weight, and will ordinarilybe at least about 50% pure, more ordinarily at least about 60% pure,generally at least about 70% pure, more generally at least about 80%pure, often at least about 85% pure, more often at least about 90% pure,preferably at least about 95% pure, more preferably at least about 98%pure, and in most preferred embodiments, at least 99% pure. Carriers orexcipients will often be added, or the formulation may be sterile orcomprise buffer components.

“Substantial similarity” in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least 56%, more generally at least 59%,ordinarily at least 62%, more ordinarily at least 65%, often at least68%, more often at least 71%, typically at least 74%, more typically atleast 77%, usually at least 80%, more usually at least about 85%,preferably at least about 90%, more preferably at least about 95 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides. Alternatively, substantial similarity exists when thesegments will hybridize under selective hybridization conditions, to astrand, or its complement, typically using a sequence derived from SEQID NO: 1 or 7, or appropriate parts of 3 and 9. Typically, selectivehybridization will occur when there is at least about 55% similarityover a stretch of at least about 30 nucleotides, preferably at leastabout 65% over a stretch of at least about 25 nucleotides, morepreferably at least about 75%, and most preferably at least about 90%over about 20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res.12:203-213. The length of similarity comparison, as described, may beover longer stretches, and in certain embodiments will be over a stretchof at least about 17 nucleotides, usually at least about 20 nucleotides,more usually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 40 nucleotides, preferably atleast about 50 nucleotides, and more preferably at least about 75 to 100or more nucleotides. The measures of comparison for the DCMP1 do notreflect on those comparison measures for the DCMP2 embodiments.

“Stringent conditions”, in referring to homology or substantialsimilarity in the hybridization context, will be stringent combinedconditions of salt, temperature, organic solvents, and other parameters,typically those controlled in hybridization reactions. The combinationof parameters is more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370. A nucleic acid probe which binds to a target nucleic acidunder stringent conditions is specific for said target nucleic acid.Such a probe is typically more than 11 nucleotides in length, and issufficiently identical or complementary to a target nucleic acid overthe region specified by the sequence of the probe to bind the targetunder stringent hybridization conditions.

Counterpart DCMP proteins from other mammalian species can be cloned andisolated by cross-species hybridization of closely related species. See,e.g., below. Similarity may be relatively low between distantly relatedspecies, and thus hybridization of relatively closely related species isadvisable. Alternatively, preparation of an antibody preparation whichexhibits less species specificity may be useful in expression cloningapproaches.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiological components. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular protein and do notsignificantly bind other proteins present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to the human DCMP1 protein immunogen with theamino acid sequence depicted in SEQ ID NO: 2 or 8 can be selected toobtain antibodies specifically immunoreactive with that DCMP protein andnot with other proteins. These antibodies recognize proteins highlysimilar to the homologous human DCMP1 protein.

III. Nucleic Acids

These DCMP genes are selectively expressed on dendritic cells. Thepreferred embodiments, as disclosed, will be useful in standardprocedures to isolate genes from other species, e.g., warm bloodedanimals, such as birds and mammals. Cross hybridization will allowisolation of related proteins from individuals, strains, or species. Anumber of different approaches are available successfully to isolate asuitable nucleic acid clone based upon the information provided herein.Southern blot hybridization studies should identify homologous genes inother species under appropriate hybridization conditions.

Purified protein or defined peptides are useful for generatingantibodies by standard methods, as described below. Synthetic peptidesor purified protein can be presented to an immune system to generatepolyclonal and monoclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, which areincorporated herein by reference. Alternatively, a DCMP antigen bindingcomposition can be useful as a specific binding reagent, and advantagecan be taken of its specificity of binding, for, e.g., purification of aDCMP protein.

The specific binding composition can be used for screening an expressionlibrary made from a cell line which expresses the respective DCMPprotein. Many methods for screening are available, e.g., standardstaining of surface expressed ligand, or by panning. Screening ofintracellular expression can also be performed by various staining orimmunofluorescence procedures. The binding compositions could be used toaffinity purify or sort out cells expressing the antigen.

TABLE 1 Alignment of primate, e.g., human, lectin/ASGPR family members.ASGPRh1 and ASGPRh2 are hepatic asialoglycoprotein receptors (see SEQ IDNO:5 and 6); ASGPRm (SEQ ID NO:12 is a macrophage derived ASGPR; DCMP2has short (SEQ ID NO:13), long (SEQ ID NOs:3 and 4), and variant forms,(SEQ ID NOs:9 and 10), DCMP1 is presented in SEQ ID NO:2 and 8. ASGPRh1MTKE..YQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGP................RLLLLSLGASGPRh2MAKD..FQDIQQLSSEENDHP.FHQGPPPAQPLAQRLCSMV................CFSLLALS ASGPRmMTRT..YENFQYLENKVKVQG.FKNGPLPLQSLLQRLRSGP................CHLLLSLG DCMP2sMTRT..YENFQYLENKVKVQG.FKNGPLPLQSLLQRLRSGP................CHLLLSLG DCMP21MTRT..YENFQYLENKVKVQG.FKNGPLPLQSLLQRLRSCP................CHLLLSLG DCMP2vMTRT..YENFQYLENKVKVQG.FKNGPLPLQS ................................ DCMP1MTSEITYAEVR...........FKNEFKSSGINTASSAASKERTAPHKSNTGFPKLLCASLLIFFfeature      ****                                                   +++++ASGPRh1LSLLLLVVVCVIGS.QNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSELSQLE.ASGPRh2FNILLLVVICVTGS.QSAQLQAELRSLKEAFSNFSSSTLTEVQAISTHGGSVGDKITSLGAKLE. ASGPRmLGLLLLVIICVVG.FQNSKFQRDLVTLRTDFSNFTSNTVAEIQALTSQGSSLEETIASLKAEVEG DCMP2sLGLLLLVIICVVG.FQNSKFQRDLVTLRTDFSNFTSNTVAEIQALTSQGSSLEETTASLKAEVEG DCMP21LGLLLLVIICVVG.FQNSKFQRDLVTLRTDFSNFTSNTVAEIQALTSQGSSLEETIASLKAEVEG DCMP2v..LLLLVIICVVG.FQNSKFQRDLVTLRTDFSNFTSNTVAEIQALTSQGSSLEETIASLKAEVEG DCMP1LLLAISFFIAFVIFFQKYS.Q..LLEKKTT.KELVHTTLE....CVKKNMPVEETAWS.......feature ++++++++ ASGPRh1.KQQK.........................DLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSASGPEh2.KQQQ.........................DLKADHDALLFHLKHFPVDLRFVACQMELLHSNGS ASGPPmFKQERQA...........................VHSENLLRVQQLVQDLKKLTCQVATLNNNGE DCMP2sFKQERQA...........................VHSEMLLRVQQLVQDLKKLTCQVATLNNN.. DCMP21FKQERQAGVSELQEHTTQKAHLGHCPHCPSVCVPVHSEMLLRVQQLVQDLKKLTCQVATLNNN.. DCMP2vFKQERQA...........................VHSENLLRVQQLVQDLKKLTCQVATLNNNGE DCMP1.................................................................feature ASGPRh1ER....TCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTASGPRh2QR....TCCPVNWVEHQGSCYWFSHSGKAWAEAEKYCQLENAHLVVINSWEEQKFIVQHTNPFNT ASGPPmEASTEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQLKNAHLVVINSREEQNFVQKYLGSAYT DCMP2s.ASTEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQLKNAHLVVINSREEQNFVQKYLGSAYT DCMP21.ASTEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQLKNAHLVVINSREEQNFVQKYLGSAYT DCMP2vEASTEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQLKNAHLVVINSREEQNFVQKYLGSAYT DCMP1.......CCPKNWKSFSSNCYFISTESASWQDSEKDCARMEAHLLVINTQEEQDFIFQNLQEESAfeature       ..........................................................ASGPPh1W.MGLHDQNGP..WKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCA..HFTDDGR...WNDDASGPRh2W.IGLTDSDGS..WKWVDGTDYRHNYKNWAVTQPDNWHGHELGGSEDCV..EVQPDGR...WNDD ASGPRmW.MGLSDPEGA..WKWVDGTDYATGFQNWKPGQPDDWQGHGLGGGEDCA..HFHPDGR...WNDD DCMP2sW.MGLSDPEGA..WKWVDGTDYATGFQNWKPGQPDNWQGHGLGGGEDCA..HFHPDGR...WNDD DCMP21W.MGLSDPEGA..WKWVDGTDYATGFQNWKPGQPDDWQGHGLGGGEDCA..HFHPDGR...WNDD DCMP2vW.MGLSDPEGA..WKWVDGTDYATGFQNWKPGQPDDWQGHGLGGGEDCA..HFHPDGR...WNDD DCMP1YFVGLSDPEGQRHWQWVDQTPYNESSTFWHPREPSD.......PNERCVVLNFRKSPKRWGWNDVfeature................................XXX..............................ASGPRh1 VCQRPYRWVCETELDKASQEPPLL ASGPRh2 FCLQVYRWVCEKRRNATGE...VA ASGPPRVCQRPYHWVCEAGLGQTSQESH DCMP2s VCQRPYHWVCEAGLGQTSQESH DCMP21VCQRPYHWVCEAGLGQTSQESH DCMP2v VCQRPYHWVCEAGLGQTSQESH DCMP1NCLGPQRSVCEMMKIH.......L feature ....................... features: ***internalization domain (an extended domain EITYAEV is seen in the NKreceptor NKA); +++ transmembrane domain; ... C-type lectin domain; XXXsugar specificity domain. The DCMPl receptor is closest in homology tothe macrophage lectin in the lectin domain.

Table 1: Alignment of primate, e.g., human, Iectin/ASGPR family members.ASGPRh1 and ASGPRh2 are hepatic asialoglycoprotein receptors (see SEQ IDNO: 5 and 6); ASGPRm (SEQ ID NO: 12) is a macrophage derived ASGPR;DCMP2 has short (SEQ ID NO: 13), long (SEQ ID NOs: 3 and 4), and variantforms (SEQ ID NOs: 9 and 10), DCMP1 is presented in SEQ ID NO: 2 and 8.

Sequence analysis suggests these DCMPs are members of thelectin/asialoglycoprotein superfamily of receptors. The peptide segmentscan also be used to design and produce appropriate oligonucleotides toscreen a library to determine the presence of a similar gene, e.g., anidentical or polymorphic variant, or to identify a DC. The genetic codecan be used to select appropriate oligonucleotides useful as probes forscreening. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingdesired clones from a library.

Complementary sequences will also be used as probes or primers. Basedupon identification of the likely amino terminus, other peptides shouldbe particularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

Techniques for nucleic acid manipulation of genes encoding these DCproteins, e.g., subcloning nucleic acid sequences encoding polypeptidesinto expression vectors, labeling probes, DNA hybridization, and thelike are described generally in Sambrook, et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, NY, which is incorporated hereinby reference and hereinafter referred to as “Sambrook, et al.” See also,Coligan, et al. (1987 and periodic supplements) Current Protocols inMolecular Biology Greene/Wiley, New York, N.Y., referred to as “Coligan,et al.”

There are various methods of isolating the DNA sequences encoding theseDC proteins. For example, DNA is isolated from a genomic or cDNA libraryusing labeled oligonucleotide probes having sequences identical orcomplementary to the sequences disclosed herein. Full-length probes maybe used, or oligonucleotide probes may be generated by comparison of thesequences disclosed with other proteins and selecting specific primers.Such probes can be used directly in hybridization assays to isolate DNAencoding DC proteins, or probes can be designed for use in amplificationtechniques such as PCR, for the isolation of DNA encoding DC proteins.

To prepare a cDNA library, mRNA is isolated from cells which express theDC protein. cDNA is prepared from the mRNA and ligated into arecombinant vector. The vector is transfected into a recombinant hostfor propagation, screening and cloning. Methods for making and screeningcDNA libraries are well known. See Gubler and Hoffman (1983) Gene25:263-269; Sambrook, et al.; or Coligan, et al.

For a genomic library, the DNA can be extracted from tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation and cloned in bacteriophage lambda vectors. These vectorsand phage are packaged in vitro, as described, e.g., in Sambrook, et al.or Coligan, et al. Recombinant phage are analyzed by plaquehybridization as described in Benton and Davis (1977) Science196:180-182. Colony hybridization is carried out as generally describedin, e.g., Grunstein, et al. (1975) Proc. Natl. Acad. Sci. USA72:3961-3965.

DNA encoding a DC protein can be identified in either cDNA or genomiclibraries by its ability to hybridize with the nucleic acid probesdescribed herein, for example in colony or plaque hybridizationexperiments. The corresponding DNA regions are isolated by standardmethods familiar to those of skill in the art. See Sambrook, et al.

Various methods of amplifying target sequences, such as the polymerasechain reaction, can also be used to prepare DNA encoding DC proteins.Polymerase chain reaction (PCR) technology is used to amplify suchnucleic acid sequences directly from mRNA, from cDNA, and from genomiclibraries or cDNA libraries. The isolated sequences encoding DC proteinsmay also be used as templates for PCR amplification.

In PCR techniques, oligonucleotide primers complementary to two 5′regions in the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers. SeeInnis, et al. (eds.) (1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif. Primers can be selectedto amplify the entire regions encoding a selected full-length DC proteinor to amplify smaller DNA segments as desired. Once such regions arePCR-amplified, they can be sequenced and oligonucleotide probes can beprepared from sequence obtained using standard techniques. These probescan then be used to isolate DNAs encoding other forms of the DCproteins.

Oligonucleotides for use as probes are chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage and Carruthers (1983) Tetrahedron Lett. 22(20):1859-1862, orusing an automated synthesizer, as described in Needham-VanDevanter, etal. (1984) Nucleic Acids Res. 12:6159-6168. Purification ofoligonucleotides is performed e.g., by native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson andRegnier (1983) J. Chrom. 255:137-149. The sequence of the syntheticoligonucleotide can be verified using the chemical degradation method ofMaxam and Gilbert in Grossman and Moldave (eds. 1980) Methods inEnzymology 65:499-560 Academic Press, New York.

This invention provides isolated DNA or fragments to encode a DCprotein, as described. In addition, this invention provides isolated orrecombinant DNA which encodes a biologically active protein orpolypeptide which is capable of hybridizing under appropriateconditions, e.g., high stringency, with the DNA sequences describedherein. Said biologically active protein or polypeptide can be anaturally occurring form, or a recombinant protein or fragment, and havean amino acid sequence as disclosed in SEQ ID NO: 2, 4, 8, or 10.Preferred embodiments will be full length natural isolates, e.g., from aprimate. In glycosylated form, the proteins should exhibit larger sizes.Further, this invention encompasses the use of isolated or recombinantDNA, or fragments thereof, which encode proteins which are homologous toeach respective DC protein. The isolated DNA can have the respectiveregulatory sequences in the 5′ and 3′ flanks, e.g., promoters,enhancers, poly-A addition signals, and others.

IV. Making DC Gene Products

DNAs which encode these DC proteins or fragments thereof can be obtainedby chemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.

These DNAs can be expressed in a wide variety of host cells for thesynthesis of a full-length protein or fragments which can, e.g., be usedto generate polyclonal or monoclonal antibodies; for binding studies;for construction and expression of modified molecules; and forstructure/function studies. Each of these DC proteins or their fragmentscan be expressed in host cells that are transformed or transfected withappropriate expression vectors. These molecules can be substantiallypurified to be free of protein or cellular contaminants, other thanthose derived from the recombinant host, and therefore are particularlyuseful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The antigen, orportions thereof, may be expressed as fusions with other proteins.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired DC gene or its fragments, usually operably linkedto suitable genetic control elements that are recognized in a suitablehost cell. These control elements are capable of effecting expressionwithin a suitable host. The specific type of control elements necessaryto effect expression will depend upon the eventual host cell used.Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently from thehost cell.

The vectors of this invention contain DNAs which encode the various DCproteins, or a fragment thereof, typically encoding, e.g., abiologically active polypeptide, or protein. The DNA can be under thecontrol of a viral promoter and can encode a selection marker. Thisinvention further contemplates use of such expression vectors which arecapable of expressing eukaryotic cDNA coding for a DC protein in aprokaryotic or eukaryotic host, where the vector is compatible with thehost and where the eukaryotic cDNA coding for the protein is insertedinto the vector such that growth of the host containing the vectorexpresses the cDNA in question. Usually, expression vectors are designedfor stable replication in their host cells or for amplification togreatly increase the total number of copies of the desirable gene percell. It is not always necessary to require that an expression vectorreplicate in a host cell, e.g., it is possible to effect transientexpression of the protein or its fragments in various hosts usingvectors that do not contain a replication origin that is recognized bythe host cell. It is also possible to use vectors that cause integrationof a DC gene or its fragments into the host DNA by recombination, or tointegrate a promoter which controls expression of an endogenous gene.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector but all other forms of vectors which servean equivalent function are suitable for use herein. See, e.g., Pouwels,et al. (1985 and Supplements) Cloning Vectors: A Laboratory ManualElsevier, N.Y.; and Rodriquez, et al. (eds.) (1988) Vectors: A Survey ofMolecular Cloning Vectors and Their Uses Buttersworth, Boston, Mass.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 or itsderivatives. Vectors that can be used to express DC proteins orfragments include, but are not limited to, such vectors as thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pp or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius, et al. (1988) “ExpressionVectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters”, inRodriguez and Denhardt (eds.) Vectors: A Survey of Molecular CloningVectors and Their Uses 10:205-236 Buttersworth, Boston, Mass.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith DC gene sequence containing vectors. For purposes of thisinvention, the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used generically to represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the desired protein or its fragments, and sequences fortranslation termination, polyadenylation, and transcription termination.Suitable expression vectors for yeast include such constitutivepromoters as 3-phosphoglycerate kinase and various other glycolyticenzyme gene promoters or such inducible promoters as the alcoholdehydrogenase 2 promoter or metallothionine promoter. Suitable vectorsinclude derivatives of the following types: self-replicating low copynumber (such as the YRp-series), self-replicating high copy number (suchas the YEp-series); integrating types (such as the YIp-series), ormini-chromosomes (such as the YCp-series).

Higher eukaryotic tissue culture cells are the preferred host cells forexpression of the DC protein. In principle, most any higher eukaryotictissue culture cell line may be used, e.g., insect baculovirusexpression systems, whether from an invertebrate or vertebrate source.However, mammalian cells are preferred to achieve proper processing,both cotranslationally and posttranslationally. Transformation ortransfection and propagation of such cells is routine. Useful cell linesinclude HeLa cells, Chinese hamster ovary (CHO) cell lines, baby ratkidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey(COS) cell lines. Expression vectors for such cell lines usually includean origin of replication, a promoter, a translation initiation site, RNAsplice sites (e.g., if genomic DNA is used), a polyadenylation site, anda transcription termination site. These vectors also may contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pCDNA1; pCD, see Okayama, et al. (1985) Mol.Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.

In certain instances, the DC proteins need not be glycosylated to elicitbiological responses in certain assays. However, it will often bedesirable to express a DC polypeptide in a system which provides aspecific or defined glycosylation pattern. In this case, the usualpattern will be that provided naturally by the expression system.However, the pattern will be modifiable by exposing the polypeptide,e.g., in unglycosylated form, to appropriate glycosylating proteinsintroduced into a heterologous expression system. For example, a DC genemay be co-transformed with one or more genes encoding mammalian or otherglycosylating enzymes. It is further understood that over glycosylationmay be detrimental to DC protein biological activity, and that one ofskill may perform routine testing to optimize the degree ofglycosylation which confers optimal biological activity.

A DC protein, or a fragment thereof, may be engineered to bephosphatidyl inositol (PI) linked to a cell membrane, but can be removedfrom membranes by treatment with a phosphatidyl inositol cleavingenzyme, e.g., phosphatidyl inositol phospholipase-C. This releases theantigen in a biologically active form, and allows purification bystandard procedures of protein chemistry. See, e.g., Low (1989) Biochem.Biophys. Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008;Brunner, et al. (1991) J. Cell Biol. 114:1275-1283; and Coligan, et al.(eds.) (1996 and periodic supplements) Current Protocols in ProteinScience, John Wiley & Sons, New York, N.Y.

Now that these DC proteins have been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis Springer-Verlag, New York, N.Y.; and Bodanszky (1984)The Principles of Peptide Synthesis Springer-Verlag, New York, N.Y. Seealso Merrified (1986) Science 232:341-347; and Dawson, et al. (1994)Science 266:776-779. For example, an azide process, an acid chlorideprocess, an acid anhydride process, a mixed anhydride process, an activeester process (for example, p-nitrophenyl ester, N-hydroxysuccinimideester, or cyanomethyl ester), a carbodiimidazole process, anoxidative-reductive process, or a dicyclohexylcarbodiimide(DCCD)/additive process can be used. Solid phase and solution phasesyntheses are both applicable to the foregoing processes.

The prepared protein and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, for example,by extraction, precipitation, electrophoresis and various forms ofchromatography, and the like. The DC proteins of this invention can beobtained in varying degrees of purity depending upon the desired use.Purification can be accomplished by use of known protein purificationtechniques or by the use of the antibodies or binding partners hereindescribed, e.g., in immunoabsorbant affinity chromatography. Thisimmunoabsorbant affinity chromatography is carried out by first linkingthe antibodies to a solid support and contacting the linked antibodieswith solubilized lysates of appropriate source cells, lysates of othercells expressing the protein, or lysates or supernatants of cellsproducing the proteins as a result of DNA techniques, see below.

Multiple cell lines may be screened for one which expresses said proteinat a high level compared with other cells. Various cell lines, e.g., amouse thymic stromal cell line TA4, is screened and selected for itsfavorable handling properties. Natural DC cell proteins can be isolatedfrom natural sources, or by expression from a transformed cell using anappropriate expression vector. Purification of the expressed protein isachieved by standard procedures, or may be combined with engineeredmeans for effective purification at high efficiency from cell lysates orsupernatants. FLAG or His₆ segments can be used for such purificationfeatures.

V. Antibodies

Antibodies can be raised to the various DC proteins, includingindividual, polymorphic, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in their recombinant forms. Additionally, antibodies can be raisedto DC proteins in either their active forms or in their inactive forms.Anti-idiotypic antibodies may also be used.

a. Antibody Production

A number of immunogens may be used to produce antibodies specificallyreactive with these DC proteins. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Naturally occurring protein may also be used either in pure or impureform. Synthetic peptides made using the human DC protein sequencesdescribed herein may also used as an immunogen for the production ofantibodies to the DC protein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described herein, and purified asdescribed. The product is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies may begenerated for subsequent use in immunoassays to measure the protein.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably a purified protein, ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to the DCprotein of interest. When appropriately high titers of antibody to theimmunogen are obtained, blood is collected from the animal and antiseraare prepared. Further fractionation of the antisera to enrich forantibodies reactive to the protein can be done if desired. See, e.g.,Harlow and Lane.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell. See, e.g., Kohler and Milstein (1976) Eur. J. Immunol.6:511-519, which is incorporated herein by reference. Alternativemethods of immortalization include transformation with Epstein BarrVirus, oncogenes, or retroviruses, or other methods known in the art.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according to thegeneral protocol outlined by Huse, et al. (1989) Science 246:1275-1281.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of these DC proteins can be raised byimmunization of animals with conjugates of the fragments with carrierproteins as described above. Monoclonal antibodies are prepared fromcells secreting the desired antibody. These antibodies can be screenedfor binding to normal or defective DC proteins, or screened foragonistic or antagonistic activity. These monoclonal antibodies willusually bind with at least a K_(D) of about 1 mM, more usually at leastabout 300 μM, typically at least about 10 μM, more typically at leastabout 30 μM, preferably at least about 10 μM, and more preferably atleast about 3 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory ManualCSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; and particularly in Kohler andMilstein (1975) Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies. Summarized briefly, this methodinvolves injecting an animal with an immunogen to initiate a humoralimmune response. The animal is then sacrificed and cells taken from itsspleen, which are then fused with myeloma cells. The result is a hybridcell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secretes a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, Huse, et al. (1989) “Generation of aLarge Combinatorial Library of the Immunoglobulin Repertoire in PhageLambda,” Science 246:1275-1281; and Ward, et al. (1989) Nature341:544-546. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al. (1989) Proc. Nat'lAcad. Sci. USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating each DC protein. Columns can be preparedwhere the antibodies are linked to a solid support, e.g., particles,such as agarose, SEPHADEX, or the like, where a cell lysate may bepassed through the column, the column washed, followed by increasingconcentrations of a mild denaturant, whereby purified DC protein will bereleased.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies to DC proteins may be used for the analysis or, oridentification of specific cell population components which express therespective protein. By assaying the expression products of cellsexpressing DC proteins it is possible to diagnose disease, e.g.,immune-compromised conditions, DC depleted conditions, or overproductionof DC.

Antibodies raised against each DC will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

b. Immunoassays

A particular protein can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites and Terr (eds.) 1991 Basic and Clinical Immunology(7th ed.). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, which are reviewedextensively in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, BocaRaton, Fla.; Tijan (1985) “Practice and Theory of Enzyme Immunoassays,”Laboratory Techniques in Biochemistry and Molecular Biology ElsevierScience Publishers B. V., Amsterdam; and Harlow and Lane Antibodies, ALaboratory Manual, supra, each of which is incorporated herein byreference. See also Chan (ed.) (1987) Immunoassay: A Practical GuideAcademic Press, Orlando, Fla.; Price and Newman (eds.) (1991) Principlesand Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988)Non-isotopic Immunoassays Plenum Press, NY.

Immunoassays for measurement of these DC proteins can be performed by avariety of methods known to those skilled in the art. In brief,immunoassays to measure the protein can be competitive or noncompetitivebinding assays. In competitive binding assays, the sample to be analyzedcompetes with a labeled analyte for specific binding sites on a captureagent bound to a solid surface. Preferably the capture agent is anantibody specifically reactive with the DC protein produced as describedabove. The concentration of labeled analyte bound to the capture agentis inversely proportional to the amount of free analyte present in thesample.

In a competitive binding immunoassay, the DC protein present in thesample competes with labeled protein for binding to a specific bindingagent, for example, an antibody specifically reactive with the DCprotein. The binding agent may be bound to a solid surface to effectseparation of bound labeled protein from the unbound labeled protein.Alternately, the competitive binding assay may be conducted in liquidphase and any of a variety of techniques known in the art may be used toseparate the bound labeled protein from the unbound labeled protein.Following separation, the amount of bound labeled protein is determined.The amount of protein present in the sample is inversely proportional tothe amount of labeled protein binding.

Alternatively, a homogeneous immunoassay may be performed in which aseparation step is not needed. In these immunoassays, the label on theprotein is altered by the binding of the protein to its specific bindingagent. This alteration in the labelled protein results in a decrease orincrease in the signal emitted by label, so that measurement of thelabel at the end of the immunoassay allows for detection or quantitationof the protein.

These DC proteins may also be quantitatively determined by a variety ofnoncompetitive immunoassay methods. For example, a two-site, solid phasesandwich immunoassay may be used. In this type of assay, a binding agentfor the protein, for example an antibody, is attached to a solidsupport. A second protein binding agent, which may also be an antibody,and which binds the protein at a different site, is labeled. Afterbinding at both sites on the protein has occurred, the unbound labeledbinding agent is removed and the amount of labeled binding agent boundto the solid phase is measured. The amount of labeled binding agentbound is directly proportional to the amount of protein in the sample.

Western blot analysis can be used to determine the presence of DCproteins in a sample. Electrophoresis is carried out, e.g., on a tissuesample suspected of containing the protein. Following electrophoresis toseparate the proteins, and transfer of the proteins to a suitable solidsupport such as a nitrocellulose filter, the solid support is incubatedwith an antibody reactive with the denatured protein. This antibody maybe labeled, or alternatively may be it may be detected by subsequentincubation with a second labeled antibody that binds the primaryantibody.

The immunoassay formats described above employ labeled assay components.The label can be in a variety of forms. The label may be coupleddirectly or indirectly to the desired component of the assay accordingto methods well known in the art. A wide variety of labels may be used.The component may be labeled by any one of several methods.Traditionally a radioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P is used. Non-radioactive labels include ligands which bind tolabeled antibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled protein. The choice of label depends on sensitivity required,ease of conjugation with the compound, stability requirements, andavailable instrumentation. For a review of various labeling or signalproducing systems which may be used, see U.S. Pat. No. 4,391,904, whichis incorporated herein by reference.

Antibodies reactive with a particular protein can also be measured by avariety of immunoassay methods. For reviews of immunological andimmunoassay procedures applicable to the measurement of antibodies byimmunoassay techniques, see, e.g., Stites and Terr (eds.) Basic andClinical Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay,supra; and Harlow and Lane Antibodies, A Laboratory Manual, supra.

A variety of different immunoassay formats, separation techniques, andlabels can be also be used similar to those described above for themeasurement of specific proteins.

VI. Purified DC Proteins

Primate, e.g., human, DCMP1 nucleotide and amino acid sequences areprovided in SEQ ID NO: 1 and 2. Rodent, e.g., mouse, DCMP1 nucleotideand amino acid seqeunces are provided in SEQ ID NO: 7 and 8. Primate,e.g., human, DCMP2 nucleotide and amino acid sequences are provided inSEQ ID NO: 3 and 4. Another variant is described in SEQ ID NO: 9 and 10.Similar primate hepatic asialoglycyprotein sequences are provided in SEQID NO: 5 and 6. The peptide sequences allow preparation of peptides togenerate antibodies to recognize such segments, and allow preparation ofoligonucleotides which encode such sequences.

VII. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence similarity with an amino acid sequence of a SEQ IDNO: 2 or 8 or selected portions of SEQ ID NO: 4 or 10. Variantsexhibiting substitutions, e.g., 20 or fewer, preferably 10 or fewer, andmore preferably 5 or fewer substitutions, are also enabled. Where thesubstitutions are conservative substitutions, the variants will shareimmunogenic or antigenic similarity or cross-reactivity with acorresponding natural sequence protein. Natural variants includeindividual, allelic, polymorphic, strain, or species variants.

Amino acid sequence similarity, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. This changes when considering conservative substitutions asmatches. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Homologousamino acid sequences include natural allelic and interspecies variationsin each respective protein sequence. Typical homologous proteins orpeptides will have from 50-100% similarity (if gaps can be introduced),to 75-100% similarity (if conservative substitutions are included) withthe amino acid sequence of the relevant DC protein. Identity measureswill be at least about 50%, generally at least 60%, more generally atleast 65%, usually at least 70%, more usually at least 75%, preferablyat least 80%, and more preferably at least 80%, and in particularlypreferred embodiments, at least 85% or more. See also Needleham, et al.(1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Time Warps,String Edits, and Macromolecules: The Theory and Practice of SequenceComparison Chapter One, Addison-Wesley, Reading, Mass.; and softwarepackages from IntelliGenetics, Mountain View, Calif.; and the Universityof Wisconsin Genetics Computer Group (GCG), Madison, Wis.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis, or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

Nucleic acids encoding the corresponding mammalian DC proteins willtypically hybridize to SEQ ID NO: 1 or 7, or appropriate portion of 3under stringent conditions. For example, nucleic acids encoding therespective DC proteins will typically hybridize to the nucleic acid ofSEQ ID NO: 1, 7, 3, or 9, under stringent hybridization conditions,while providing few false positive hybridization signals. Generally,stringent conditions are selected to be about 10° C. lower than thethermal melting point (Tm) for the sequence being hybridized to at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa perfectly matched probe. Typically, stringent conditions will be thosein which the salt concentration in wash is about 0.02 molar at pH 7 andthe temperature is at least about 50° C. Other factors may significantlyaffect the stringency of hybridization, including, among others, basecomposition and size of the complementary strands, the presence oforganic solvents such as formamide, and the extent of base mismatching.A preferred embodiment will include nucleic acids which will bind todisclosed sequences in 50% formamide and 20-50 mM NaCl at 42° C.Hybridization under stringent conditions should give a background of atleast 2-fold over background, preferably at least 3-5 or more.

An isolated DC gene DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode these DC antigens, their derivatives, orproteins having highly similar physiological, immunogenic, or antigenicactivity.

Modified sequences can be used to produce mutant antigens or to enhanceexpression. Enhanced expression may involve gene amplification,increased transcription, increased translation, and other mechanisms.Such mutant DC protein derivatives include predetermined orsite-specific mutations of the respective protein or its fragments.“Mutant DC protein” encompasses a polypeptide otherwise falling withinthe homology definition of the DC protein as set forth above, but havingan amino acid sequence which differs from that of the DC protein asfound in nature, whether by way of deletion, substitution, or insertion.In particular, “site specific mutant DC protein” generally includesproteins having significant similarity with a protein having a sequenceof SEQ ID NO: 2 or 8. Generally, the variant will share manyphysicochemical and biological activities, e.g., antigenic orimmunogenic, with those sequences, and in preferred embodiments containmost or all of the disclosed sequence. Similar concepts apply to thesevarious DC proteins, particularly those found in various warm bloodedanimals, e.g., primates and mammals.

Although site specific mutation sites are predetermined, mutants neednot be site specific. DC protein mutagenesis can be conducted by makingamino acid insertions or deletions. Substitutions, deletions,insertions, or any combinations may be generated to arrive at a finalconstruct. Insertions include amino- or carboxyl-terminal fusions.Random mutagenesis can be conducted at a target codon and the expressedmutants can then be screened for the desired activity. Methods formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known in the art, e.g., by M13 primermutagenesis or polymerase chain reaction (PCR) techniques. See also,Sambrook, et al. (1989) and Ausubel, et al. (1987 and Supplements). Themutations in the DNA normally should not place coding sequences out ofreading frames and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structure such as loops orhairpins.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of an immunoglobulin with a respective DC polypeptide is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties derived from each source peptide. A similar concept appliesto heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, domains or othersegments may be “swapped” between different new fusion polypeptides orfragments, typically with related proteins, e.g., withn the lectin orasialoglycoprotein families. Preferably, intact structural domains willbe used, e.g., intact Ig portions. See, e.g., Cunningham, et al. (1989)Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem.263:15985-15992. Thus, new chimeric polypeptides exhibiting newcombinations of specificities will result from the functional linkage ofprotein-binding specificities and other functional domains. Also,alanine scanning mutagenesis may be applied, preferably to residueswhich structurally are exterior to the secondary structure, which willavoid most of the critical residues which generally disrupt tertiarystructure.

“Derivatives” of these DC antigens include amino acid sequence mutants,glycosylation variants, and covalent or aggregate conjugates with otherchemical moieties. Covalent derivatives can be prepared by linkage offunctionalities to groups which are found in these DC protein amino acidside chains or at the N- or C-termini, by means which are well known inthe art. These derivatives can include, without limitation, aliphaticesters or amides of the carboxyl terminus, or of residues containingcarboxyl side chains, O-acyl derivatives of hydroxyl group-containingresidues, and N-acyl derivatives of the amino terminal amino acid oramino-group containing residues, e.g., lysine or arginine. Acyl groupsare selected from the group of alkyl-moieties including C3 to C18 normalalkyl, thereby forming alkanoyl aroyl species. Covalent attachment tocarrier proteins may be important when immunogenic moieties are haptens.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine, or other moieties, including ribosylgroups or cross-linking reagents. Also, proteins comprisingsubstitutions are encompassed, which should retain substantialimmunogenicity, to produce antibodies which recognize a protein of SEQID NO: 2, 4, 8, or 10. Typically, these proteins will contain less than20 residue substitutions from the disclosed sequence, more typicallyless than 10 substitutions, preferably less than 5, and more preferablyless than three. Alternatively, proteins which begin and end atstructural domains will usually retain antigenicity and crossimmunogenicity.

A major group of derivatives are covalent conjugates of the DC proteinsor fragments thereof with other proteins or polypeptides. Thesederivatives can be synthesized in recombinant culture such as N- orC-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred protein derivatization sites with cross-linking agents are atfree amino groups, carbohydrate moieties, and cysteine residues.

Fusion polypeptides between these DC proteins and other homologous orheterologous proteins are also provided. Heterologous polypeptides maybe fusions between different surface markers, resulting in, e.g., ahybrid protein. Likewise, heterologous fusions may be constructed whichwould exhibit a combination of properties or activities of thederivative proteins. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused protein may be easily determined. See, e.g., Dull, et al.,U.S. Pat. No. 4,859,609. Other gene fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, and yeast alpha mating factor. See, e.g., Godowski, etal. (1988) Science 241:812-816.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

This invention also contemplates the use of derivatives of these DCproteins other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into the threeclasses: (1) salts, (2) side chain and terminal residue covalentmodifications, and (3) adsorption complexes, for example with cellmembranes. Such covalent or aggregative derivatives are useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of ligands or other binding ligands. Forexample, a DC protein antigen can be immobilized by covalent bonding toa solid support such as cyanogen bromide-activated Sepharose, by methodswhich are well known in the art, or adsorbed onto polyolefin surfaces,with or without glutaraldehyde cross-linking, for use in the assay orpurification of anti-DC protein antibodies. The DC proteins can also belabeled with a detectable group, e.g., radioiodinated by the chloramineT procedure, covalently bound to rare earth chelates, or conjugated toanother fluorescent moiety for use in diagnostic assays. Purification ofthese DC proteins may be effected by immobilized antibodies.

Isolated DC protein genes will allow transformation of cells lackingexpression of a corresponding DC protein, e.g., either species types orcells which lack corresponding proteins and exhibit negative backgroundactivity. Expression of transformed genes will allow isolation ofantigenically pure cell lines, with defined or single specie variants.This approach will allow for more sensitive detection and discriminationof the physiological effects of these DC proteins. Subcellularfragments, e.g., cytoplasts or membrane fragments, can be isolated andused.

VIII. Binding Agent:DC Protein Complexes

A DC protein that specifically binds to or that is specificallyimmunoreactive with an antibody generated against a defined immunogen,such as an immunogen consisting of the amino acid sequence of SEQ ID NO:2, 4, 8, or 10, is determined in an immunoassay. The immunoassay uses apolyclonal antiserum which was raised to the protein of SEQ ID NO: 2, 4,8, or 10. This antiserum is selected to have low crossreactivity againstother members of the related families, and any such crossreactivity isremoved by immunoabsorbtion prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO: 2, 4, 8, or 10, is isolated as described herein. For example,recombinant protein may be produced in a mammalian cell line. An inbredstrain of mice such as balb/c is immunized with the appropriate proteinusing a standard adjuvant, such as Freund's adjuvant, and a standardmouse immunization protocol (see Harlow and Lane, supra). Alternatively,a synthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Polyclonalsera are collected and titered against the immunogen protein in animmunoassay, e.g., a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross reactivity againstother related proteins, using a competitive binding immunoassay such asthe one described in Harlow and Lane, supra, at pages 570-573.Preferably two different related protiens are used in this determinationin conjunction with a given DC protein. For example, with the lectinprotein, at least two other family members are used to absorb out sharedepitopes. In conjunction with the DCMP1 family member, two other membersof the family are used. These other family members can be produced asrecombinant proteins and isolated using standard molecular biology andprotein chemistry techniques as described herein.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO: 2or 8 can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the protein of SEQ IDNO 2 or 8. The percent crossreactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% crossreactivity with each of the proteins listed above are selectedand pooled. The cross-reacting antibodies are then removed from thepooled antisera by immunoabsorbtion with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the DC protein of SEQ ID NO: 2 or 8). Inorder to make this comparison, the two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required is less thantwice the amount of the protein of SEQ ID NO: 2 or 8 that is required,then the second protein is said to specifically bind to an antibodygenerated to the immunogen.

It is understood that DC proteins are likely a family of homologousproteins that comprise two or more genes. For a particular gene product,such as the human Ig family member protein, the invention encompassesnot only the amino acid sequences disclosed herein, but also to otherproteins that are allelic, polymorphic, non-allelic, or speciesvariants. It also understood that the term “human DC protein” includesnonnatural mutations introduced by deliberate mutation usingconventional recombinant technology such as single site mutation, or byexcising short sections of DNA encoding these proteins or splicevariants from the gene, or by substituting or adding samll numbers ofnew amino acids. Such minor alterations must substantially maintain theimmunoidentity of the original molecule and/or its biological activity.Thus, these alterations include proteins that are specificallyimmunoreactive with a designated naturally occurring respective DCprotein, e.g., the human DC protein exhibiting SEQ ID NO: 4. Particularprotein modifications considered minor would include conservativesubstitution of amino acids with similar chemical properties, asdescribed above for each protein family as a whole. By aligning aprotein optimally with the protein of SEQ ID NO 2 or 8, and by using theconventional immunoassays described herein to determine immunoidentity,one can determine the protein compositions of the invention.

IX. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for developmental abnormalities, or below in thedescription of kits for diagnosis. In particular, the genes will beuseful as markers for distinguishing cell types, including genomicaspects of cells, as well as mRNA and protein expression patterns.

DC genes, e.g., DNA or RNA may be used as a component in a forensicassay. For instance, the nucleotide sequences provided may be labeledusing, e.g., ³²P or biotin and used to probe standard restrictionfragment polymorphism blots, providing a measurable character to aid indistinguishing between individuals. Such probes may be used inwell-known forensic techniques such as genetic fingerprinting. Inaddition, nucleotide probes made from DC sequences may be used in insitu assays to detect chromosomal abnormalities.

Antibodies and other binding agents directed towards DC proteins ornucleic acids may be used to purify the corresponding DC proteinmolecule. As described in the Examples below, antibody purification ofDC proteins is both possible and practicable. Antibodies and otherbinding agents may also be used in a diagnostic fashion to determinewhether DC components are present in a tissue sample or cell populationusing well-known techniques described herein. The ability to attach abinding agent to a DC protein provides a means to diagnose disordersassociated with expression misregulation. Antibodies and other DCprotein binding agents may also be useful as histological or forensicmarkers. As described in the examples below, the expression of each ofthese proteins is limited to specific tissue types. By directing aprobe, such as an antibody or nucleic acid to the respective DC protein,it is possible to use the probe to distinguish tissue and cell types insitu or in vitro.

This invention also provides reagents which may exhibit significanttherapeutic value. The DC proteins (naturally occurring or recombinant),fragments thereof, and antibodies thereto, along with compoundsidentified as having binding affinity to the DC protein, may be usefulin the treatment of conditions associated with abnormal physiology ordevelopment, including abnormal proliferation, e.g., cancerousconditions, or degenerative conditions. Abnormal proliferation,regeneration, degeneration, and atrophy may be modulated by appropriatetherapeutic treatment using the compositions provided herein. Forexample, a disease or disorder associated with abnormal expression orabnormal signaling by a DC, e.g., as an antigen presenting cell, is atarget for an agonist or antagonist of the protein. The proteins likelyplay a role in regulation or development of hematopoietic cells, e.g.,lymphoid cells, which affect immunological responses, e.g., antigenpresentation and the resulting effector functions.

Other abnormal developmental conditions are known in cell types shown topossess DC protein mRNA by northern blot analysis. See Berkow (ed.) TheMerck Manual of Diagnosis and Therapy. Merck & Co., Rahway, N.J.; andThorn, et al. Harrison's Principles of Internal Medicine, McGraw-Hill,N.Y. Developmental or functional abnormalities, e.g., of the immunesystem, cause significant medical abnormalities and conditions which maybe susceptible to prevention or treatment using compositions providedherein.

Recombinant DC proteins or antibodies might be purified and thenadministered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inconventional pharmaceutically acceptable carriers or diluents, e.g.,immunogenic adjuvants, along with physiologically innocuous stabilizersand excipients. In particular, these may be useful in a vaccine context,where the antigen is combined with one of these therapeutic versions ofagonists or antagonists. These combinations can be sterile filtered andplaced into dosage forms as by lyophilization in dosage vials or storagein stabilized aqueous preparations. This invention also contemplates useof antibodies or binding fragments thereof, including forms which arenot complement binding.

Drug screening using antibodies or receptor or fragments thereof canidentify compounds having binding affinity to these DC proteins,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicstimulating activity and is therefore a blocker or antagonist in that itblocks the activity of the protein. Likewise, a compound havingintrinsic stimulating activity might activate the cell through theprotein and is thus an agonist in that it simulates the cell. Thisinvention further contemplates the therapeutic use of antibodies to theproteins as antagonists.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

The DC proteins, fragments thereof, and antibodies to it or itsfragments, antagonists, and agonists, could be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8thed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosgae Forms: TabletsDekker, N.Y.; and Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Disperse Systems Dekker, N.Y. The therapy of this invention maybe combined with or used in association with other chemotherapeutic orchemopreventive agents.

Both the naturally occurring and the recombinant form of the DC proteinsof this invention are particularly useful in kits and assay methodswhich are capable of screening compounds for binding activity to theproteins. Several methods of automating assays have been developed inrecent years so as to permit screening of tens of thousands of compoundsin a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773,and other descriptions of chemical diversity libraries, which describemeans for testing of binding affinity by a plurality of compounds. Thedevelopment of suitable assays can be greatly facilitated by theavailability of large amounts of purified, e.g., soluble versions of, DCprotein as provided by this invention.

For example, antagonists can often be found once the protein has beenstructurally defined. Testing of potential protein analogs is nowpossible upon the development of highly automated assay methods using apurified surface protein. In particular, new agonists and antagonistswill be discovered by using screening techniques described herein. Ofparticular importance are compounds found to have a combined bindingaffinity for multiple related cell surface antigens, e.g., compoundswhich can serve as antagonists for species variants of a DC protein.

This invention is particularly useful for screening compounds by usingrecombinant DC protein in a variety of drug screening techniques. Theadvantages of using a recombinant protein in screening for specificligands include: (a) improved renewable source of the protein from aspecific source; (b) potentially greater number of antigens per cellgiving better signal to noise ratio in assays; and (c) species variantspecificity (theoretically giving greater biological and diseasespecificity).

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing a DC protein. Cells may be isolated which express thatprotein in isolation from any others. Such cells, either in viable orfixed form, can be used for standard surface protein binding assays. Seealso, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells (source of DC protein) arecontacted and incubated with an antibody having known binding affinityto the antigen, such as ¹²⁵I-antibody, and a test sample whose bindingaffinity to the binding composition is being measured. The bound andfree labeled binding compositions are then separated to assess thedegree of protein binding. The amount of test compound bound isinversely proportional to the amount of labeled antibody binding to theknown source. Many techniques can be used to separate bound from freereagent to assess the degree of binding. This separation step couldtypically involve a procedure such as adhesion to filters followed bywashing, adhesion to plastic followed by washing, or centrifugation ofthe cell membranes Viable cells could also be used to screen for theeffects of drugs on these DC protein mediated functions, e.g., antigenpresentation or helper function.

Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of a DC protein. These cells arestably transformed with DNA vectors directing the expression of thesppropriate protein, e.g., an engineered membrane bound form.Essentially, the membranes would be prepared from the cells and used inbinding assays such as the competitive assay set forth above.

Still another approach is to use solubilized, unpurified or solubilized,purified DC protein from transformed eukaryotic or prokaryotic hostcells. This allows for a “molecular” binding assay with the advantagesof increased specificity, the ability to automate, and high drug testthroughput.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto the respective DC protein and is described in detail in Geysen,European Patent Application 84/03564, published on Sep. 13, 1984. First,large numbers of different small peptide test compounds are synthesizedon a solid substrate, e.g., plastic pins or some other appropriatesurface, see Fodor, et al., supra. Then all the pins are reacted withsolubilized, unpurified or solubilized, purified DC protein, and washed.The next step involves detecting bound reagent, e.g., antibody.

One means for determining which sites interact with specific otherproteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions.For a detailed description of protein structural determination, see,e.g., Blundell and Johnson (1976) Protein Crystallography AcademicPress, NY.

X. Kits

This invention also contemplates use of these DC proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of a DC protein or message.Typically the kit will have a compartment containing either a defined DCpeptide or gene segment or a reagent which recognizes one or the other,e.g., antibodies.

A kit for determining the binding affinity of a test compound to therespective DC protein would typically comprise a test compound; alabeled compound, for example an antibody having known binding affinityfor the protein; a source of the DC protein (naturally occurring orrecombinant); and a means for separating bound from free labeledcompound, such as a solid phase for immobilizing the DC protein. Oncecompounds are screened, those having suitable binding affinity to theprotein can be evaluated in suitable biological assays, as are wellknown in the art, to determine whether they act as agonists orantagonists to regulate DC function. The availability of recombinant DCpolypeptides also provide well defined standards for calibrating suchassays.

A preferred kit for determining the concentration of, for example, a DCprotein in a sample would typically comprise a labeled compound, e.g.,antibody, having known binding affinity for the DC protein, a source ofDC protein (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, for example, a solidphase for immobilizing the DC protein. Compartments containing reagents,and instructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for therespective DC or its fragments are useful in diagnostic applications todetect the presence of elevated levels of the protein and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescence, cell cultures, body fluids, and further caninvolve the detection of antigens in serum, or the like. Diagnosticassays may be homogeneous (without a separation step between freereagent and antigen-DC protein complex) or heterogeneous (with aseparation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), enzymeimmunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT),substrate-labeled fluorescent immunoassay (SLFIA), and the like. Forexample, unlabeled antibodies can be employed by using a second antibodywhich is labeled and which recognizes the antibody to the DC protein orto a particular fragment thereof. Similar assays have also beenextensively discussed in the literature. See, e.g., Harlow and Lane(1988) Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987)Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price andNewman (eds.) (1991) Principles and Practice of Immunoassay StocktonPress, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press,NY. In particular, the reagents may be useful for diagnosing DCpopulations in biological samples, either to detect an excess ordeficiency of DC in a sample. The assay may be directed to histologicalanalysis of a biopsy, or evaluation of DC numbers in a blood or tissuesample.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a DC protein, as such may be diagnostic of variousabnormal states. For example, overproduction of the DC protein mayresult in various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or receptor, or labeled DC proteinis provided. This is usually in conjunction with other additives, suchas buffers, stabilizers, materials necessary for signal production suchas substrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In many of these assays, the protein, testcompound, DC protein, or antibodies thereto can be labeled eitherdirectly or indirectly. Possibilities for direct labeling include labelgroups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) suchas peroxidase and alkaline phosphatase, and fluorescent labels (U.S.Pat. No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the freeprotein, or alternatively the bound from the free test compound. The DCprotein can be immobilized on various matrices followed by washing.Suitable matrices include plastic such as an ELISA plate, filters, andbeads. Methods of immobilizing the DC protein to a matrix include,without limitation, direct adhesion to plastic, use of a captureantibody, chemical coupling, and biotin-avidin. The last step in thisapproach involves the precipitation of protein/antibody complex by oneof several methods including those utilizing, e.g., an organic solventsuch as polyethylene glycol or a salt such as ammonium sulfate. Othersuitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magneticparticle separation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of arespective DC protein. These sequences can be used as probes fordetecting levels of the message in samples from patients suspected ofhaving an abnormal condition, e.g., cancer or immune problem. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²P. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorophores, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in any conventional techniques such as nucleicacid hybridization, plus and minus screening, recombinational probing,hybrid released translation (HRT), and hybrid arrested translation(HART). This also includes amplification techniques such as polymerasechain reaction (PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

XI. Binding Partner Isolation

Having isolated one member of a binding partner of a specificinteraction, methods exist for isolating the counter-partner. See,Gearing, et al. (1989) EMBO J. 8:3667-3676. For example, means to labela DC surface protein without interfering with the binding to itsreceptor can be determined. For example, an affinity label can be fusedto either the amino- or carboxyl-terminus of the ligand. An expressionlibrary can be screened for specific binding to the DC protein, e.g., bycell sorting, or other screening to detect subpopulations which expresssuch a binding component. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad.Sci. USA 90:11267-11271. Alternatively, a panning method may be used.See, e.g., Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA84:3365-3369. A two-hybrid selection system may also be applied makingappropriate constructs with the available DC protein sequences. See,e.g., Fields and Song (1989) Nature 340:245-246.

Protein cross-linking techniques with label can be applied to isolatebinding partners of a DC protein. This would allow identification ofproteins which specifically interact with the appropriate DC protein.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention tospecific embodiments.

EXAMPLES

General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSHPress, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, NY.

Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan, et al. (1996 and periodic Supplements) CurrentProtocols in Protein Science Wiley/Greene, NY; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments, e.g., to aFLAG sequence or an equivalent which can be fused via aprotease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, NY; and Crowe, et al.(1992) QIAexpress: The High Level Expression & Protein PurificationSystem QUIAGEN, Inc., Chatsworth, Calif.

Methods for determining immunological function are described, e.g., inColigan, et al. (1992 and periodic Supplements) Current Protocols inImmunology Wiley/Greene, NY See also, e.g., Paul (ed.) (1993)Fundamental Immunology (3d ed.) Raven Press, N.Y.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Generation of Dendritic Cells

Human CD34+ cells were obtained as follows. See, e.g., Caux, et al.(1995) pages 1-5 in Banchereau and Schmitt Dendritic Cells inFundamental and Clinical Immunology Plenum Press, NY. Peripheral or cordblood cells, sometimes CD34+ selected, were cultured in the presence ofStem Cell Factor (SCF), GM-CSF, and TNF-a in endotoxin free RPMI 1640medium (GIBCO, Grand Island, N.Y.) supplemented with 10% (v/v)heat-inactivated fetal bovine serum (FBS; Flow Laboratories, Irvine,Calif.), 10 mM HEPES, 2 mM L-glutamine, 5×10⁻⁵ M 2-mercaptoethanol,penicillin (100 mg/ml). This is referred to as complete medium.

CD34+ cells were seeded for expansion in 25 to 75 cm² flasks (Corning,N.Y.) at 2×10⁴ cells/ml. Optimal conditions were maintained by splittingthese cultures at day 5 and 10 with medium containing fresh GM-CSF andTNF-a (cell concentration: 1-3×10⁵ cells/ml). In certain cases, cellswere FACS sorted for CD1a expression at about day 6.

In certain situations, cells were routinely collected after 12 days ofculture, eventually adherent cells were recovered using a 5 mM EDTAsolution. In other situations, the CD1a+ cells were activated byresuspension in complete medium at 5×10⁶ cells/ml and activated for theappropriate time (e.g., 1 or 6 h) with 1 mg/ml phorbol 12-myristate13-acetate (PMA, Sigma) and 100 ng/ml ionomycin (Calbiochem, La Jolla,Calif.). These cells were expanded for another 6 days, and RNA isolatedfor cDNA library preparation.

III. RNA Isolation and Library Construction

Total RNA is isolated using, e.g., the guanidine thiocyanate/CsClgradient procedure as described by Chirgwin, et al. (1978) Biochem.18:5294-5299.

Alternatively, poly(A)+ RNA is isolated using the OLIGOTEX mRNAisolation kit (QIAGEN). Double stranded cDNA are generated using, e.g.,the SUPERSCRIPT plasmid system (Gibco BRL, Gaithersburg, Md.) for cDNAsynthesis and plasmid cloning. The resulting double stranded cDNA isunidirectionally cloned, e.g., into psportl and transfected byelectroporation into ELECTROMAX DH10BTM Cells (Gibco BRL, Gaithersburg,Md.).

IV. Sequencing

DNA isolated from randomly picked clones, or after subtractivehybridization using unactivated cells, were subjected to nucleotidesequence analysis using standard techniques. A Taq DiDeoxy Terminatorcycle sequencing kit (Applied Biosystems, Foster City, Calif.) can beused. The labeled DNA fragments are separated using a DNA sequencing gelof an appropriate automated sequencer. Alternatively, the isolated cloneis sequenced as described, e.g., in Maniatis, et al. (1982) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor Press; Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual, (2d ed.), vols 1-3, CSH Press, NY; Ausubel, et al., Biology,Greene Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987and Supplements) Current Protocols in Molecular Biology, Greene/Wiley,New York. Chemical sequencing methods are also available, e.g., usingMaxam and Gilbert sequencing techniques.

V. Recombinant DC Gene Construct

Poly(A)⁺ RNA is isolated from appropriate cell populations, e.g., usingthe FastTrack mRNA kit (Invitrogen, San Diego, Calif.). Samples areelectrophoresed, e.g., in a 1% agarose gel containing formaldehyde andtransferred to a GeneScreen membrane (NEN Research Products, Boston,Mass.). Hybridization is performed, e.g., at 65° C. in 0.5 M NaHPO₄ pH7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V) with ³²P-dCTP labeled DCgene cDNA at 10⁷ cpm/ml. After hybridization filters are washed threetimes at 50° C. in 0.2×SSC, 0.1% SDS, and exposed to film for 24 h.

The recombinant gene construct may be used to generate a probe fordetecting the message. The insert may be excised and used in thedetection methods described above.

VI. Expression of DC Gene Protein in E. coli

PCR is used to make a construct comprising the open reading frame,preferably in operable association with proper promoter, selection, andregulatory sequences. The resulting expression plasmid is transformedinto an appropriate, e.g., the Topp5, E. coli strain (Stratagene, LaJolla, Calif.). Ampicillin resistant (50 μg/ml) transformants are grownin Luria Broth (Gibco) at 37° C. until the optical density at 550 nm is0.7. Recombinant protein is induced with 0.4 mMisopropyl-bD-thiogalacto-pyranoside (Sigma, St. Louis, Mo.) andincubation of the cells continued at 20° C. for a further 18 hours.Cells from a 1 liter culture are harvested by centrifugation andresuspended, e.g., in 200 ml of ice cold 30% sucrose, 50 mM Tris HCl pH8.0, 1 mM ethylenediaminetetraacetic acid. After 10 min on ice, ice coldwater is added to a total volume of 2 liters. After 20 min on ice, cellsare removed by centrifugation and the supernatant is clarified byfiltration via a 5 μM Millipak 60 (Millipore Corp., Bedford, Mass.).

The recombinant protein is purified via standard purification methods,e.g., various ion echange chromatography methods. Immunoaffinity methodsusing antibodies described below can also be used. Affinity methods maybe used where an epitope tag is engineered into an expression construct.

VII. Mapping of Human DC Genes

DNA isolation, restriction enzyme digestion, agarose gelelectrophoresis, Southern blot transfer and hybridization are performedaccording to standard techniques. See Jenkins, et al. (1982) J. Virol.43:26-36. Blots may be prepared with Hybond-N nylon membrane (Amersham).The probe is labeled with ³²P-dCTP; washing is done to a finalstringency, e.g., of 0.1×SSC, 0.1% SDS, 65° C.

Alternatively, a BIOS Laboratories (New Haven, Conn.) mouse somatic cellhybrid panel may be combined with PCR methods. See Fan, et al. (1996)Immunogenetics 44:97-103.

Chromosomal localization with a Stanford G3 panel gave as closest markerSHGC-12041, with a lod of 7.7. This marker, which is the gene coding forM130 antigen, is localized to chromosome 12p13. This localization ishost to a number of genes encoding receptors of the C-type lectinfamily, notably CD69, and the NK receptor family.

VIII. Analysis of Individual Variation

From the distribution data, an abundant easily accessible cell type isselected for sampling from individuals. Using PCR techniques, a largepopulation of individuals are analysed for this gene. cDNA or other PCRmethods are used to sequence the corresponding gene in the differentindividuals, and their sequences are compared. This indicates both theextent of divergence among racial or other populations, as well asdetermining which residues are likely to be modifiable without dramaticeffects on function.

IX. Preparation of Antibodies

Recombinant DC proteins are generated by expression in E. coli as shownabove, and tested for biological activity. Alternatively, naturalprotein sources may be used with purification methods made available.Antibody reagents may be used in immunopurification, or to trackseparation methods. Active or denatured proteins may be used forimmunization of appropriate mammals for either polyclonal serumproduction, or for monoclonal antibody production.

X. Isolation of Counterpart Primate or Rodent DC Genes

Human cDNA clones encoding these genes are used as probes, or to designPCR primers to find counterparts in various primate species, e.g.,chimpanzees.

Bioinformatics searches of the EST databases (GenBank dbEST) using thepredicted polypeptide sequence of DCMP1 (tblastn algorithm) revealedmouse clones encoding a protein homologous to primate DCMP1. Four clonescorresponding to this sequence were seen: AA387662 Ko mouse embryo 115dpc; AA170532 mouse spleen; AA475012 mouse mammary gland; and AA423158mouse mammary gland. One of these, AA170532, estimated to be a fulllength clone by sequence analysis was selected and DNA sequenced. Thisclone contained features similar to DCMP1. The full length clone is 1418bp, excluding the poly-A sequence and contains a 5′ UTR of 278 bp. Asfor hDCMP1, the putative start codon is not contained within a consensusKozak region, but this codon is preceeded by an upstream stop codon. The5′ UTR contains sequences similar to rapid degradation signals,including three consensus ATTTA sites. A potential polyadenylationsequence is seen. The predicted polypeptide is about 238 residues inlength and codes for a type II membrane protein with an ITIM and aC-type lectin domain. Three potential N-glycosylation sites are seen.Alignments of this and the human protein show about 54% identity, 65%homology over the whole sequence. Notably, the ITIM domains are highlyconserved (13 out of 15 residues are identical). Of interest is theconserved membrane-proximal glutamine motif (FQKYSQLLE), and thecysteine residue potentially implicated in disulphide bridge formation.Equally the C-type lectin domains show blocks of conservation, includingthe EPS motif. Differences seen between hDCMP1 and the human hepaticlectins are retained in the mouse sequence, notably the replacement oftryptophan at position 119 and 163; a glutamine at position 177; andserine instead of tryptophan at position 228. It thus appears that thisclone is the mouse homologue of hDCMP1.

XI. Use of Reagents to Analyze Cell Populations

Detection of the level of dendritic cells present in a a sample isimportant for diagnosis of aberrant disease conditions. For example, anincrease in the number of dendritic cells in a tissue or the lymphsystem can be indicative of the presence of a DC hyperplasia, or tissueor graft rejection. A low DC population can indicate an abnormalreaction to, e.g., a bacterial or viral infection, which may require theappropriate treat to normalize the DC response.

FACS analysis using a labeled binding agent specific for a cell surfaceDC protein, see, e.g., Melamed, et al. (1990) Flow Cytometry and SortingWiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y., is used indetermining the number of DCs present in a cell mixture, e.g., PBMCs,adherent cells, etc. The binding agent is also used for histologicalanalysis of tissue samples, either fresh or fixed, to analyzeinfiltration of DC. Diverse cell populations may also be evaluated,either in a cell destructive assay, or in certain assays where cellsretain viability.

Analysis of the presence of soluble intracellular molecules isperformed, e.g., with a fluorescent binding agent specific for a DC asdescribed in Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367.Alternatively, tissue or cell fixation methods may be used.

Levels of DC transcripts are quantitated, e.g., using semiquantitativePCR as described in Murphy, et al. (1993) J. Immunol. Methods162:211-223. Primers are designed such that genomic DNA is not detected.

XII. Expression Distribution

Analysis of the entire DCMP1 cDNA sequence in a sequence databaserevealed an expression pattern restricted to a limited number oflibraries. The greatest number of sequences (ten) were detected inDendritic Cell libraries, four sequences in a library of osteoclastomacells, and single sequences from libraries of macrophages generated invitro from monocytes, LPS activated neutrophils, chondrosarcoma, coloncancer, T-cell lymphoma, skin tumor and chronic synovitis. In theGenBank dbEST, four clones were detected: AA418441, subtracted library;AA446401, total fetus; AA677149, fetal liver spleen; C01555, Human GeneSignature; AA380065, Skin tumor.

Analysis of DCMP1 expression by RT-PCR over a number of different celllines and freshly isolated cells showed that expression of DCMP1 is notdetected in TF1 (Myeloid precursor), Jurkat (a T cell line), CHA (kidneycarcinoma), MRC5 (fetal lung fibroblasts), JY (B cell line), U937(myelo-monocytic lymphoma cell line), but is restricted tohaematopoeitic cells. In freshly isolated cells, expression is seen inDC, both non-activated and activated; granulocytes activated; PBL, bothnon-activated and activated; and a low level of expression is seen inmonocytes activated; and B cells activated. All activated samples werepools of cells treated with PMA/ionomycin for 1 and 6 hours.

Additional analysis showed that expression of DCMP1 varied with theactivation state of the cell. RT-PCR was also used to detect theexpression of DCMP1 under different activation states. B cells isolatedfrom tonsillar tissue were treated with PMA/ionomycin for 1 or 6 hoursor by coculture with CD40L-expressing L cells for 3, 12, and 24 hours.mRNA was detected in non-activated cells. This expression could be lostwithin 1 hour for PMA/ionomycin treatment and after 3 hours of CD40Ltreatment. In contrast, no expression could be detected in T cells, evenafter anti-CD3/anti-CD28 incubation.

In CD34+ derived cells expression of DCMP1 was strong in macrophagesderived from CD34+ progenitor cells in the presence of M-CSF. Thisexpression did not appear to alter in response to PMA/ionomycin. In DCderived from CD34+ progenitor cells in the presence of GM-CSF and TNFa,the level of mRNA was seen to vary over the time course of culture, withgreater amounts of mRNA detected at day 12 of culture. After 48 hours ofcoculture with CD40L bearing L cells, the expression of DCMP1 is lost.In in vitro DC FACS sorted at day 6 for the presence of markersCD1a/CD14 and continued in culture for a further 6 days, more mRNA wasdetected in the CD14 than in the CD1a subpopulation. This expression wasdown-regulated by PMA/ionomycin treatment.

In monocytes isolated from blood, no mRNA was detected in non-activatedcells. However, expression of DCMP1 was detected after 6 hours oftreatment with PMA/ionomycin. In DC derived from monocytes by treatmentwith GM-CSF and IL-4, DCMP1 expression was upregulated. This expressioncould not be altered by treatment with PMA/ionomycin, but could bedownregulated by coculture with CD40L expressing L cells. In this caseDCMP1 mRNA expression was totally lost by 24 hours of culture.Expression of the human protein was cinfirmed using antibody detectionmethods.

DCMP1 was expressed in subsets of DC isolated ex vivo. DC subsetsisolated from blood or from tonsillar tissue were characterized by thepresence or absence of the integrin CD11c. Larson and Springer (1990)Immunol. Rev. 114:181-217. The CD11c+ subset of DC isolated from blood(also known as GCDC) express DCMP1. However, no mRNA is detected afteractivation via an anti-CD40L or PMA/ionomycin treatment. In contrast,the same subset of cells isolated from tonsillar tissue no longerexpress DCMP1. In the case of the CD11c-DC subset, a low level ofexpression is observed in cells isolated from blood. This expression isgreater in cells isolated from tonsillar tissue, but again isdownregulated on activation via an anti-CD40 antibody or withPMA/ionomycin treatment. Langerhans cells isolated from skin expressDCMP1, while the surrounding basal cells show no expression.

XIII. Primate DCMP1

Sequence analysis suggests these DCMPs are members of thelectin/asialoglycoprotein superfamily of receptors. In particular, theheapatic and macrophage lectins have been associated with theinternalization of proteins and peptides, which, e.g., might beimportant in the uptake and presentation of antigen by dendritic cells.The DCMP1 contains an internalization motif (YxxV) or an ITIM-like motif(IxYxxV; residues 5-10 of SEQ ID NO: 2; a more extended motif runs fromresidues 1 to 24). This suggests that the protein may be a dendriticcell version of the family of Inhibitory Receptors (KIR; LIR, etc.),which send a negative signal to inhibit cell function.

The putative open reading frame commences at about nucleotide 242. Thispotential start codon is not in a consensus Kozak sequence, but since itis not preceeded by an alternative ATG and a stop codon exists atupstream position 200, it is predicted that this is the start of theencoded protein. A polypeptide of about 237 amino acids was predictedfrom this sequence. No signal peptide was detected, but a putativetransmembrane sequence extends from positions about 386 to 443. Thisclone encodes a type II membrane protein with a C-type lectin domain.The 3′ UTR contains a number of potential rapid degradation signals,including three repeats of the consensus sequence ATTTA. No signalpeptide was detected, but putative transmembrane sequences extend frompositions 45 to 62, or alternatively, 386 to 443. This clone encodes atype II membrane protein with a C-type lectin domain.

The polypeptide predicted from the sequence analysis has a 49 amino acidintracellular domain which includes a tyrosine-based motif centred atresidue 7. YXX(L/V) motifs of this nature have been shown to act asinternalization motifs in the case of the hepatic lectins and CD23 (FceRIIa). This type of domain has been shown to act as activation (ITAM) orinhibitory (ITIM) motifs in molecules such as Ly49, NKG2A, and the KIRfamily of immunoglobulin-like molecules. Inhibition is mediated by therecruitment of SHP2/SHIP phosphatases to the consensus domain(I/V)XYXX(L/V). The first 15 amino acids of DCMP1 show conservation tothe extended ITIM domain, and it seems likely that inhibition of cellfunction is one of the attributes of DCMP1. A single potentialN-glycosylation site is present at about position 185.

Comparison of the amino acid sequence of the C-type lectin domain ofDCMP1 with other proteins containing C-type lectin domains showed thatDCMP1 has the greatest homology to the hepatic lectins and themacrophage lectin (see Table 1). The conserved cysteine residues of theC-type lectin fold are clearly conserved across the members of thisfamily, however a number of distingishing features can be seen. Like thehepatic lectins, DCMP1 has a double cysteine motif at the start of thelectin domain. The function of this supplementary cysteine is unknown asthere is apparently no other cysteine in the lectin domain that may forma disulphide bridge with this residue. It is possible that this residuemay be involved in intermolecular disulphide bridge formation, althoughthere is another cysteine in DCMP1 at position 91 which probably fulfilsthis function. The N-terminal portion of the DCMP1 lectin domain showsgreatest conservation with the hepatic lectins and the macrophagelectin. The calcium-binding domain is conserved in DCMP1 and showsgreatest homology to CD23, including the EPS motif (residues 195-197),glutamate (E) at position 201 and asparagine aspartate (ND) at position218-219. These motifs are noticeably absent from the NK receptors (NKGEshown here) and CD94.

The DCMP1 is a type II membrane protein with the predicted transmembranesegment from about residues 45 to 62. It is related to the family ofproteins which includes asialoglycoprotein receptors, hepatic lectins,CD69, CD72, CD23, and NK receptors. This protein contains anextracellular Ca dependent C-type lectin domain at the carboxy terminus(from about residues 104 to 237), which exhibits the motifscharacteristic of sugar residue specificity. See Table 1. These proteinstypically bind to sugar residues on glycoproteins and are implicated inthe primary immune response. Several members of this family, notablyCD69, the NK receptors, and CD72, have been shown to transmit a signalduring cellular activation events including proliferation and theinduction of specific genes.

DCMP1, like the mouse C-type lectin KIR receptor, Ly 49, contains aninternalisation motif with extended homology to the group of inhibitoryreceptors (ITIM domain, see recent reviews by Vivier and Daeron (1997)Immunology Today 18:286-291; and Katz and Austen (1997) J. Immunol.158:5065-5070). These receptors, either Immunoglobulin superfamily(IgSF) members or C-type lectins, transmit a negative signal viaSH2-domain containing phosphatases, e.g., SHIP, SHP-1, and SHP-2.Evidence suggests that these receptors associate with other activationreceptors in order to block activation signals. Evidence also suggeststhat the ligand for this type of molecule is an IgSF molecule. Examplesof this are the MHC class I molecules (recognised by the CD94/NKreceptors and Ly-49) and the FcgR (CD23; which recognises IgG).

The cysteine residue 91 is likely to be involved in disulfide linkage toanother polypeptide, perhaps a homo or heterodimer.

PCR analysis indicates that the gene is expressed in activated dendriticcells and non-activated dendritic cells. Detectable signals were notfound in any of TF1 (hematopoietic cell line), Jurkat (T cell line),MRC5 (lung fibroblast sarcoma cell line), JY (B cell line), U937(pre-monocyte cell line), or CHA (carcinoma cell line) cells. Positivesignals were detected in freshly isolated activated or non-activatedPBLs, and granulocytes, but only weak signals from freshly isolated Tcells, B cells, NK cells, or monocytes.

Sequence analysis indicates expression of the gene in samplescharacterized as dendritic cells, activated neutrophils, macrophages(activated with GM-CSF), osteoclastoma, skin tumor, T-cell lymphoma,colon cancer, chronic synovitis, and chrondrosarcoma.

XIV. Rodent Counterpart DCMP1

Table 2 shows sequence of rodent counterpart sequences.

TABLE 2 The sequence shows homology to two ESTs of mouse, W33446 (seeSEQ ID NO:11) and AA170532 (see SEQ ID NO:8) which code for the mousecounterpart of DCMP1 (see SEQ ID NO:8). Also shown is hDCMP1 (SEQ IDNO:2). hDCMP1 MTSEITYAEVRFKNEFKSSGINTASSAASKERTAPLKSNTGFPKLLCASL W33446-------------------------------------------------- 170532MASEITYAEVKFKNESNSLHTYSESPAAPREKPIRDLRKPGSPSLLLTSL mDCMP1MASEITYAEVKFKNESNSLHTYSESPAAPREKPIRDLRKPGSPSLLLTSL hDCMP1LIFFLLLAISFFIAFVIFFQKYSQLLE-KKTTKELVHTTLECVKKNMPVE W33446--------------------------E-KMIIKELNYTELECTKWASLLE 170532MLLLLLLAITFLVAFIIYFQKYSQLLEEKKAAKNIM mDCMP1MLLLLLLAITFLVAFIIYFQKYSQLLEEKKAAKNIMHNELNCTKSVSPME hDCMP1ETAWSCCPKNWKSFSSNCYFISTE--SASWQDSEKDCARMEAHLLVINTQ w33446DKVWSCCPKDWKPFGSYCYFTSTD-LVASWNESKENCFHMGAHLVVIHSQ mDCMP1DKVWSCCPKDWRLFGSHCYLVPTVSSSASWNKSEENCSRNGAHLVVIQSQ hDCMP1EEQDFIFQNLQEESAYFVGLSDPEGQRHWQWVDQTPYNESSTFWHPREPS W33446 EEQ mDCMP1EEQDFITGILDTHAAYFIGLWD-TGHRQWQWVDQTPYEESITFWHNGEPS hDCMP1DPNERCVVLNFR-KSPKRWGWNDVNCLGPQRSVCEMMKIHL mDCMP1SGNEKCATIIYRWKT--GWGWNDISCSLKQKSVCPMKKINL

Table 2: The sequence shows homology to two ESTs of mouse, W33446 (seeSEQ ID NO: 11) and AA170532 (see SEQ ID NO: 8) which code for the mousecounterpart of DCMP1 (See SEQ ID NO: 8). Also shown is hDCMP1 (SEQ IDNO: 2).

XV. Primate DCMP2

DCMP2, a putative asialoglycoprotein receptor, is a type IItransmembrane protein. In its extracellular region, DCMP2 features asingle carbohydrate recognition domain (CRD), characteristic of theC-type (Ca++ dependent) family of lectins (see Drickamer and Taylor(1993) Ann. Rev. Cell Biol. 9:237-264. DCMP2 displays considerablehomology with the two genes (H1 and H2) encoding the subunits of thehuman hepatic asialoglycoprotein-receptor. Stockert (1995) Physiol.Revs. 75:591-609. These hepatic receptors represent the prototype of thetype II C-type lectin family members. Liver ASGPR has bindingspecificity for desialylated glycoproteins displaying terminalgalactosyl residues, and mediates their endocytosis into hepatocytes viathe clathrin-coated pit pathway. Notably, the features associated withboth these functions are conserved between the hepatic ASGPR and DCMP2.Thus, DCMP2 contains an intracellular motif including a tyrosine residueat position 5 and which is associated with ligand endocytosis capacity.See Fuhrer, et al. (1991) J. Cell Biol. 114:423-431. In addition, theDCMP2 display a QPD (Gln-Pro-Asp) galactose-recognition type sequence(Drickamer (1992) Nature 360:183-186) in its sugar recognition domain.

Several variant cDNA clones encoding the DCMP2 have been isolated, mostlikely as a consequence of alternative splicing. Three variants aredescribed hereunder: a short form, a long form, and a third formdesignated DCMP2v. See SEQ ID NO: 4 and 10; Table 1. The short and longforms differ by the presence of a unique 27 aa insert in theextracellular region of the short form clone. The short form of theDCMP2 exhibits 4 residue differences in the extracellular region to arecently cloned ASGPR obtained from human macrophages (M-ASGPR). Suzuki,et al. (1996) J. Immunol. 156:128-135.

Relative to the DCMP21, the ASGPRm lacks the seqment corresponding toGVSELQEHTTQKAHLGHCPHCPSVCVP (residues 118-144 of SEQ ID NO: 4), and theASGPRm contains an insert of GEE (between residues 173 and 174 of SEQ IDNO: 4). The DCMP2s is identical to the DCMP21, except for the absence ofthe GVSELQEHTTQKAHLGHCPHCPSVCVP (residues 118-144 of SEQ ID NO: 4), anda difference in sequence at nucleotide 1064 from G to A, therebyencoding asn rather than asp. The DCMPv is similar to DCMPs, but lacksthe sequence LLQRLRSGPCHLLLSLGLG (residues 30-48 of SEQ ID NO: 4), whichcorresponds to a significant portion of the transmembrane segment; andcontains the insert of GEE (between residues 173 and 174 of SEQ ID NO:4) as found in the ASGPRm. Regions surrounding these differences, e.g.,within an epitope length, e.g., 12-17 amino acids, are of interest.

Recombinant DCMP2 long form protein is available, and mABs have beengenerated. In addition, a murine cell line has been transfected forstable expression of both the long and short forms.

The gene was originally isolated from 70% pure CD1a⁺ DC derived fromCD34⁺ hematopoietic progenitor cells cultured in GM-CSF and TNFa (Caux,et al. (1992) Nature 360:258-261. The clone has been inserted into apSport1 vector (NotI/SalI restriction sites).

PCR analysis suggests expression of DCMP2 genes in dendritic cells, andperhaps very weakly in TF1 (hematopoietic cell line) cells. There wasnot detectable signal from Jurkat (T cell line), CHA (carcinoma cellline), MRC5 (lung fibroblast sarcoma cell line), or JY (B cell line).Signal was detected in freshly isolated non-activated or activated (PMAand ionomycin) dendritic cells, granulocytes, and non-activated oractivated PBL. Signal was not detected in monocytes, non-activated oractivated T cells, or non-activated or activated B cells.

DC-ASPGR displays considerable homology with the murine counterpart ofhuman monocyte ASGPR (M-ASGPR). Homology is striking (˜60%) within thecarbohydrate-recognition domain which confers specificity to murinemonocyte ASGPR for galactose and N-acetylgalactosamine (GalNAc). Sato,et al. (1992) J. Biochem. 111:331-336. This includes the QPD motif, alsofound in the H1 and H2 subunits of the hepatic ASGPR. In addition,murine monocyte ASGPR has a YENL internalization signal in its cytosolicdomain.

Murine M-ASGPR functions as a receptor for endocytosis of galactosylatedglycoproteins (Ozaki, et al. (1992) J. Biol. Chem. 267:9229-9235), andallows recognition of malignant cells by tumoricidal macrophages(Kawakami, et al. (1994) Jpn. J. Cancer Res. 85:744-749). In thiscontext, murine M-ASGPR was found to be expressed within lung metastaticnodules of mice bearing OV2944-HM-1 metastatic ovarian tumor cells(Imai, et al. (1995) Immunol. 86:591-598). Of interest, human M-ASGPRdemonstrates a remarkable specificity for Tn antigen (Suzuki, et al.(1996) J. Immunol. 156:128-135), which bears a cluster of serine orthreonine-linked terminal GalNAc, and is associated with humancarcinomas (Springer (1989) Mol. Immunol. 26:1-5; and Ørntoft, et al.(1990) Int. J. Cancer 45:666-672).

On the basis of sequence homology, it can be predicted that DCMPs alsofunction as an endocytic receptor for galactosylated glycoproteins. Inaddition, ligand internalization via the mannose-receptor, anotherC-type transmembrane endocytic lectin, results in highly efficientantigen-presentation by DC through the MHC class II pathway. Cella, etal. (1997) Current Opinion Immunol. 9:10-16. By analogy, it is possiblethat the DCMPs play a similar role in routing internalized ligands intoan antigen-presentation pathway.

Thus, DCMP2 could be a potential high-efficiency target for loadingantigens into DC for enhancing presentation to T cells in immune-basedadjuvant therapy. This could be approached by pulsing DC in vitro eitherwith a galactosylated form of antigen, or with anti-DCMP2 mAbs coupledto antigen. In vitro efficiency of presentation could be assayed byactivation of antigen-specific T cells. This would focus on presentationof tumor-associated antigens (TAA), due to the inherent therapeuticperspectives of such an approach. Of particular interest are TAAassociated with malignant melanoma.

In addition, the specificity of human M-ASGPR for Tn antigen makes thiscarcinoma TAA a candidate of choice for targeting the DCMP2.

As has been recently shown that exogenous antigen can be processed andpresented in the MHC class I pathway. See Porgador and Gilboa (1995) J.Exp. Med. 182:255-260; Paglia, et al. (1996) J. Exp. Med. 183:317-322.Specialized receptors are likely to perform such a function in DC.

These receptors in DC may be targetted to help produce TAA-specificcytotoxic T cells (CTL), with significant therapeutic potential, as CTLappear to be implicated in the induction of tumor rejection.

XVI. DCMP Internalization

DC obtained from CD34+ progenitors cultured in GM-CSF and TNFa werestained at 4° C. with anti-DCMP2 mAb, or anti-CD13 as control. Followingsubsequent incubation at 37° C. for a period of up to about 20 min, cellsurface bound mAbs were analyzed. Internalization was observed bydecrease in cell surface fluorescence.

The DCMP21 is rapidly internalized at 37° C., but not at 40° C. About60% of the surface label disappeared within about 15 min. Thisdemonstrates that the DCMP2 can function as an endocytic receptor,consistent with the presence of an internalization motif (YENF) in itsintracytoplasmic domain.

XVII. Isolation of a Binding Counterpart

A DC protein can be used as a specific binding reagent, by takingadvantage of its specificity of binding, much like an antibody would beused. A binding reagent is either labeled as described above, e.g.,fluorescence or otherwise, or immobilized to a substrate for panningmethods.

The DC protein is used to screen for a cell line which exhibits binding.Standard staining techniques are used to detect or sort intracellular orsurface expressed ligand, or surface expressing transformed cells arescreened by panning. Screening of intracellular expression is performedby various staining or immunofluorescence procedures. See also McMahan,et al. (1991) EMBO J. 10:2821-2832.

For example, on day 0, precoat 2-chamber permanox slides with 1 ml perchamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature.Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cells per chamberin 1.5 ml of growth media. Incubate overnight at 37° C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 mg/mlDEAE-dextran, 66 mM chloroquine, and 4 mg DNA in serum free DME. Foreach set, a positive control is prepared, e.g., of human receptor-FLAGcDNA at 1 and {fraction (1/200)} dilution, and a negative mock. Rinsecells with serum free DME. Add the DNA solution and incubate 5 hr at 37°C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Removeand wash once with DME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed andstained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin(0.1%) with 32 ml/ml of 1M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add protein or protein/antibodycomplex to cells and incubate for 30 min. Wash cells twice withHBSS/saponin. If appropriate, add first antibody for 30 min. Add secondantibody, e.g., Vector anti-mouse antibody, at {fraction (1/200)}dilution, and incubate for 30 min. Prepare ELISA solution, e.g., VectorElite ABC horseradish peroxidase solution, and preincubate for 30 min.Use, e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin)per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRPsolution and incubate for 30 min. Wash cells twice with HBSS, secondwash for 2 min, which closes cells. Then add Vector diaminobenzoic acid(DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2drops of H₂O₂ per 5 ml of glass distilled water. Carefully removechamber and rinse slide in water. Air dry for a few minutes, then add 1drop of Crystal Mount and a cover slip. Bake for 5 min at 85-90° C.

Alternatively, other monocyte protein specific binding reagents are usedto affinity purify or sort out cells expressing a receptor. See, e.g.,Sambrook, et al. or Ausubel, et al.

Another strategy is to screen for a membrane bound receptor by panning.The receptor cDNA is constructed as described above. The ligand can beimmobilized and used to immobilize expressing cells. Immobilization maybe achieved by use of appropriate antibodies which recognize, e.g., aFLAG sequence of a monocyte protein fusion construct, or by use ofantibodies raised against the first antibodies. Recursive cycles ofselection and amplification lead to enrichment of appropriate clones andeventual isolation of ligand expressing clones.

Phage expression libraries can be screened by monocyte protein.Appropriate label techniques, e.g., anti-FLAG antibodies, will allowspecific labeling of appropriate clones.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

11 1 1104 DNA Unknown mammalian nucleic acid 1 ttctcactat actggtcctgaggaaagggc ttctgtgaac tgcggttttt agtttttatt 60 gtggttctta gttctcatgagacccctctt gaggatatgt gcctatctgg tgcctctgct 120 ctccactagt tgagtgaaaggaaggaggta atttaccacc atgtttggtt cctgtttata 180 agatgtttta agaaagatttgaaacagatt ttctgaagaa agcagaagct ctcttcccat 240 tatgacttcg gaaatcacttatgctgaagt gaggttcaaa aatgaattca agtcctcagg 300 catcaacaca gcctcttctgcagcttccaa ggagaggact gcccctctca aaagtaatac 360 cggattcccc aagctgctttgtgcctcact gttgatattt ttcctgctat tggcaatctc 420 attctttatt gcttttgtcattttctttca aaaatattct cagcttcttg aaaaaaagac 480 tacaaaagag ctggttcatacaacattgga gtgtgtgaaa aaaaatatgc ccgtggaaga 540 gacagcctgg agctgttgcccaaagaattg gaagtcattt agttccaact gctactttat 600 ttctactgaa tcagcatcttggcaagacag tgagaaggac tgtgctagaa tggaggctca 660 cctgctggtg ataaacactcaagaagagca ggatttcatc ttccagaatc tgcaagaaga 720 atctgcttat tttgtggggctctcagatcc agaaggtcag cgacattggc aatgggttga 780 tcagacacca tacaatgaaagttccacatt ctggcatcca cgtgagccca gtgatcccaa 840 tgagcgctgc gttgtgctaaattttcgtaa atcacccaaa agatggggct ggaatgatgt 900 taattgtctt ggtcctcaaaggtcagtttg tgagatgatg aagatccact tatgaactga 960 acattctcca tgaacaggtggttggattgg tatctgtcat tgtagggata gataataagc 1020 tcttcttatt catgtgtaagggaggtccat agaatttagg tggtctgtca actattctac 1080 ttatgagaga attggtctgtacat 1104 2 237 PRT Unknown mammalian protein 2 Met Thr Ser Glu Ile ThrTyr Ala Glu Val Arg Phe Lys Asn Glu Phe 1 5 10 15 Lys Ser Ser Gly IleAsn Thr Ala Ser Ser Ala Ala Ser Lys Glu Arg 20 25 30 Thr Ala Pro Leu LysSer Asn Thr Gly Phe Pro Lys Leu Leu Cys Ala 35 40 45 Ser Leu Leu Ile PhePhe Leu Leu Leu Ala Ile Ser Phe Phe Ile Ala 50 55 60 Phe Val Ile Phe PheGln Lys Tyr Ser Gln Leu Leu Glu Lys Lys Thr 65 70 75 80 Thr Lys Glu LeuVal His Thr Thr Leu Glu Cys Val Lys Lys Asn Met 85 90 95 Pro Val Glu GluThr Ala Trp Ser Cys Cys Pro Lys Asn Trp Lys Ser 100 105 110 Phe Ser SerAsn Cys Tyr Phe Ile Ser Thr Glu Ser Ala Ser Trp Gln 115 120 125 Asp SerGlu Lys Asp Cys Ala Arg Met Glu Ala His Leu Leu Val Ile 130 135 140 AsnThr Gln Glu Glu Gln Asp Phe Ile Phe Gln Asn Leu Gln Glu Glu 145 150 155160 Ser Ala Tyr Phe Val Gly Leu Ser Asp Pro Glu Gly Gln Arg His Trp 165170 175 Gln Trp Val Asp Gln Thr Pro Tyr Asn Glu Ser Ser Thr Phe Trp His180 185 190 Pro Arg Glu Pro Ser Asp Pro Asn Glu Arg Cys Val Val Leu AsnPhe 195 200 205 Arg Lys Ser Pro Lys Arg Trp Gly Trp Asn Asp Val Asn CysLeu Gly 210 215 220 Pro Gln Arg Ser Val Cys Glu Met Met Lys Ile His Leu225 230 235 3 1458 DNA Unknown mammalian nucleic acid 3 gttgaggagatgggatgtcc cagatgatag ggctcctggg atttcagacc caagaccagc 60 aggactccagtcacctctac cccagctctc caggacacag cgctcccaac tctgagtgac 120 gtcccacctctggtccttgc agcacaacca acgtgggaat cacaccctcc agacctccca 180 cagctccaccccagactggg cgccggccct gcctccattt cagctgtgac aacctcagag 240 ccgtgttggcccaagcatga caaggacgta tgaaaacttc cagtacttgg agaataaggt 300 gaaagtccaggggtttaaaa atgggccact tcctctccag tccctcctgc agcgtctccg 360 ctctgggccctgccatctcc tgctgtccct gggcctcggc ctgctgctgc tggtcatcat 420 ctgtgtggttggattccaaa attccaaatt tcagagggac ctggtgaccc tgagaacaga 480 ttttagcaacttcacctcaa acactgtggc ggagatccag gcactgactt cccagggcag 540 cagcttggaagaaacgatag catctctgaa agctgaggtg gagggtttca agcaggaacg 600 gcaggcaggggtatctgagc tccaggaaca cactacgcag aaggcacacc taggccactg 660 tccccactgcccatctgtgt gtgtcccagt tcattctgaa atgctcctgc gagtccagca 720 gctggtgcaagacctgaaga aactgacctg ccaggtggct actctcaaca acaatgcctc 780 cactgaagggacctgctgcc ccgtcaactg ggtggagcac caagacagct gctactggtt 840 ctctcactctgggatgtcct gggccgaggc tgagaagtac tgccagctga agaacgccca 900 cctggtggtcatcaactcca gggaggagca gaattttgtc cagaaatatc taggctccgc 960 atacacctggatgggcctca gtgaccctga aggagcctgg aagtgggtgg atggaacaga 1020 ctatgcgaccggcttccaga actggaagcc aggccagcca gacgactggc aggggcacgg 1080 gctgggtggaggcgaggact gtgctcactt ccatccagac ggcaggtgga atgacgacgt 1140 ctgccagaggccctaccact gggtctgcga ggctggcctg ggtcagacca gccaggagag 1200 tcactgagctgcctttggtg ggaccacccg gccacagaaa tggcggtggg aggaggactc 1260 ttctcacgacctcctcgcaa gaccgctctg ggagagaaat aagcactggg agattggaag 1320 cactgctaacattttgaatt tttttctctt taattttaaa aagatggtat agtgttctta 1380 agcttttattttttttccaa cttttgaaag tcaacttcat gaaggtataa tttttacata 1440 ataaaaatgcactcattt 1458 4 316 PRT Unknown mammalian protein 4 Met Thr Arg Thr TyrGlu Asn Phe Gln Tyr Leu Glu Asn Lys Val Lys 1 5 10 15 Val Gln Gly PheLys Asn Gly Pro Leu Pro Leu Gln Ser Leu Leu Gln 20 25 30 Arg Leu Arg SerGly Pro Cys His Leu Leu Leu Ser Leu Gly Leu Gly 35 40 45 Leu Leu Leu LeuVal Ile Ile Cys Val Val Gly Phe Gln Asn Ser Lys 50 55 60 Phe Gln Arg AspLeu Val Thr Leu Arg Thr Asp Phe Ser Asn Phe Thr 65 70 75 80 Ser Asn ThrVal Ala Glu Ile Gln Ala Leu Thr Ser Gln Gly Ser Ser 85 90 95 Leu Glu GluThr Ile Ala Ser Leu Lys Ala Glu Val Glu Gly Phe Lys 100 105 110 Gln GluArg Gln Ala Gly Val Ser Glu Leu Gln Glu His Thr Thr Gln 115 120 125 LysAla His Leu Gly His Cys Pro His Cys Pro Ser Val Cys Val Pro 130 135 140Val His Ser Glu Met Leu Leu Arg Val Gln Gln Leu Val Gln Asp Leu 145 150155 160 Lys Lys Leu Thr Cys Gln Val Ala Thr Leu Asn Asn Asn Ala Ser Thr165 170 175 Glu Gly Thr Cys Cys Pro Val Asn Trp Val Glu His Gln Asp SerCys 180 185 190 Tyr Trp Phe Ser His Ser Gly Met Ser Trp Ala Glu Ala GluLys Tyr 195 200 205 Cys Gln Leu Lys Asn Ala His Leu Val Val Ile Asn SerArg Glu Glu 210 215 220 Gln Asn Phe Val Gln Lys Tyr Leu Gly Ser Ala TyrThr Trp Met Gly 225 230 235 240 Leu Ser Asp Pro Glu Gly Ala Trp Lys TrpVal Asp Gly Thr Asp Tyr 245 250 255 Ala Thr Gly Phe Gln Asn Trp Lys ProGly Gln Pro Asp Asp Trp Gln 260 265 270 Gly His Gly Leu Gly Gly Gly GluAsp Cys Ala His Phe His Pro Asp 275 280 285 Gly Arg Trp Asn Asp Asp ValCys Gln Arg Pro Tyr His Trp Val Cys 290 295 300 Glu Ala Gly Leu Gly GlnThr Ser Gln Glu Ser His 305 310 315 5 291 PRT Unknown mammalian protein5 Met Thr Lys Glu Tyr Gln Asp Leu Gln His Leu Asp Asn Glu Glu Ser 1 5 1015 Asp His His Gln Leu Arg Lys Gly Pro Pro Pro Pro Gln Pro Leu Leu 20 2530 Gln Arg Leu Cys Ser Gly Pro Arg Leu Leu Leu Leu Ser Leu Gly Leu 35 4045 Ser Leu Leu Leu Leu Val Val Val Cys Val Ile Gly Ser Gln Asn Ser 50 5560 Gln Leu Gln Glu Glu Leu Arg Gly Leu Arg Glu Thr Phe Ser Asn Phe 65 7075 80 Thr Ala Ser Thr Glu Ala Gln Val Lys Gly Leu Ser Thr Gln Gly Gly 8590 95 Asn Val Gly Arg Lys Met Lys Ser Leu Glu Ser Gln Leu Glu Lys Gln100 105 110 Gln Lys Asp Leu Ser Glu Asp His Ser Ser Leu Leu Leu His ValLys 115 120 125 Gln Phe Val Ser Asp Leu Arg Ser Leu Ser Cys Gln Met AlaAla Leu 130 135 140 Gln Gly Asn Gly Ser Glu Arg Thr Cys Cys Pro Val AsnTrp Val Glu 145 150 155 160 His Glu Arg Ser Cys Tyr Trp Phe Ser Arg SerGly Lys Ala Trp Ala 165 170 175 Asp Ala Asp Asn Tyr Cys Arg Leu Glu AspAla His Leu Val Val Val 180 185 190 Thr Ser Trp Glu Glu Gln Lys Phe ValGln His His Ile Gly Pro Val 195 200 205 Asn Thr Trp Met Gly Leu His AspGln Asn Gly Pro Trp Lys Trp Val 210 215 220 Asp Gly Thr Asp Tyr Glu ThrGly Phe Lys Asn Trp Arg Pro Glu Gln 225 230 235 240 Pro Asp Asp Trp TyrGly His Gly Leu Gly Gly Gly Glu Asp Cys Ala 245 250 255 His Phe Thr AspAsp Gly Arg Trp Asn Asp Asp Val Cys Gln Arg Pro 260 265 270 Tyr Arg TrpVal Cys Glu Thr Glu Leu Asp Lys Ala Ser Gln Glu Pro 275 280 285 Pro LeuLeu 290 6 287 PRT Unknown mammalian protein 6 Met Ala Lys Asp Phe GlnAsp Ile Gln Gln Leu Ser Ser Glu Glu Asn 1 5 10 15 Asp His Pro Phe HisGln Gly Pro Pro Pro Ala Gln Pro Leu Ala Gln 20 25 30 Arg Leu Cys Ser MetVal Cys Phe Ser Leu Leu Ala Leu Ser Phe Asn 35 40 45 Ile Leu Leu Leu ValVal Ile Cys Val Thr Gly Ser Gln Ser Ala Gln 50 55 60 Leu Gln Ala Glu LeuArg Ser Leu Lys Glu Ala Phe Ser Asn Phe Ser 65 70 75 80 Ser Ser Thr LeuThr Glu Val Gln Ala Ile Ser Thr His Gly Gly Ser 85 90 95 Val Gly Asp LysIle Thr Ser Leu Gly Ala Lys Leu Glu Lys Gln Gln 100 105 110 Gln Asp LeuLys Ala Asp His Asp Ala Leu Leu Phe His Leu Lys His 115 120 125 Phe ProVal Asp Leu Arg Phe Val Ala Cys Gln Met Glu Leu Leu His 130 135 140 SerAsn Gly Ser Gln Arg Thr Cys Cys Pro Val Asn Trp Val Glu His 145 150 155160 Gln Gly Ser Cys Tyr Trp Phe Ser His Ser Gly Lys Ala Trp Ala Glu 165170 175 Ala Glu Lys Tyr Cys Gln Leu Glu Asn Ala His Leu Val Val Ile Asn180 185 190 Ser Trp Glu Glu Gln Lys Phe Ile Val Gln His Thr Asn Pro PheAsn 195 200 205 Thr Trp Ile Gly Leu Thr Asp Ser Asp Gly Ser Trp Lys TrpVal Asp 210 215 220 Gly Thr Asp Tyr Arg His Asn Tyr Lys Asn Trp Ala ValThr Gln Pro 225 230 235 240 Asp Asn Trp His Gly His Glu Leu Gly Gly SerGlu Asp Cys Val Glu 245 250 255 Val Gln Pro Asp Gly Arg Trp Asn Asp AspPhe Cys Leu Gln Val Tyr 260 265 270 Arg Trp Val Cys Glu Lys Arg Arg AsnAla Thr Gly Glu Val Ala 275 280 285 7 1418 DNA unknown mammalian nucleicacid 7 ctatccccca ctttgcagta cttgcatatc ttgctgagtg ggtttgaggg ctacaattct60 tattttctta tgttaagagg ttgcatttcc cttatctcgc cctggtgatt ctatgctgtg 120gtttcttgtt ctcatctcgt ttatcctagt gagacatgtc tcttctttca tacaactgtg 180caatatgaca acttatcaca gtgattggtt ctcatatact atagagcctt agagaaggaa 240caaggctctc ttctgacgga ggaagatttt ttcttgatat ggcttcagaa atcacttatg 300cagaagtgaa gttcaagaat gaatccaact ccttgcacac ctactcagaa tctcctgcag 360ctcccagaga gaaacctatc cgtgatctaa gaaagcctgg ttccccctca ctgcttctta 420catccctgat gctacttctc ctgctgctgg caatcacatt cttagttgct tttatcattt 480attttcaaaa gtactctcaa cttcttgaag aaaaaaaagc tgcaaaaaat ataatgcaca 540atgaattgaa ctgcacaaaa agtgtttcac ccatggaaga caaagtctgg agctgttgcc 600caaaggattg gaggctattt ggttcccact gctacttggt tcccacagtt tcttcatcag 660catcttggaa caagagtgag gagaactgct cccgcatggg tgctcatcta gtggtgatcc 720aaagccagga agagcaggat ttcatcactg ggatcttgga cactcatgct gcttatttta 780tagggttgtg ggatacaggc catcggcaat ggcaatgggt tgatcagaca ccatatgaag 840aaagtatcac attctggcac aatggtgagc ccagcagtgg caatgaaaaa tgtgctacaa 900taatttaccg ttggaagact ggatggggct ggaacgatat ctcttgcagt cttaaacaga 960agtcagtttg tcagatgaag aaaataaact tatgaatcac tcattcttca tgggcattcg 1020attcattgtt atccaaccat tacacagaca cctgggaaat tctacaggtt cacagaattt 1080aagtgggcag caaatggtta tgcatacact ggcccacata tatccttgtg catttaccca 1140cctactctgt cataaaatga actttcattg agaattttct atataccaca gagtatacag 1200agtcccttat ggacacacat ggaacttttt gccatcttgt ttactcatgc cattgtatga 1260taggttctct tgacctatct gtttctgttt ctctgttgtt tttttaatgt ctttggattt 1320attgacatta aattgagaag taaaattata aatatttaag tgtctggatt gatacacaca 1380gatatgtact atgaaatata attaaatatt tactgtcc 1418 8 238 PRT unknownmammalian protein 8 Met Ala Ser Glu Ile Thr Tyr Ala Glu Val Lys Phe LysAsn Glu Ser 1 5 10 15 Asn Ser Leu His Thr Tyr Ser Glu Ser Pro Ala AlaPro Arg Glu Lys 20 25 30 Pro Ile Arg Asp Leu Arg Lys Pro Gly Ser Pro SerLeu Leu Leu Thr 35 40 45 Ser Leu Met Leu Leu Leu Leu Leu Leu Ala Ile ThrPhe Leu Val Ala 50 55 60 Phe Ile Ile Tyr Phe Gln Lys Tyr Ser Gln Leu LeuGlu Glu Lys Lys 65 70 75 80 Ala Ala Lys Asn Ile Met His Asn Glu Leu AsnCys Thr Lys Ser Val 85 90 95 Ser Pro Met Glu Asp Lys Val Trp Ser Cys CysPro Lys Asp Trp Arg 100 105 110 Leu Phe Gly Ser His Cys Tyr Leu Val ProThr Val Ser Ser Ser Ala 115 120 125 Ser Trp Asn Lys Ser Glu Glu Asn CysSer Arg Met Gly Ala His Leu 130 135 140 Val Val Ile Gln Ser Gln Glu GluGln Asp Phe Ile Thr Gly Ile Leu 145 150 155 160 Asp Thr His Ala Ala TyrPhe Ile Gly Leu Trp Asp Thr Gly His Arg 165 170 175 Gln Trp Gln Trp ValAsp Gln Thr Pro Tyr Glu Glu Ser Ile Thr Phe 180 185 190 Trp His Asn GlyGlu Pro Ser Ser Gly Asn Glu Lys Cys Ala Thr Ile 195 200 205 Ile Tyr ArgTrp Lys Thr Gly Trp Gly Trp Asn Asp Ile Ser Cys Ser 210 215 220 Leu LysGln Lys Ser Val Cys Gln Met Lys Lys Ile Asn Leu 225 230 235 9 1370 DNAunknown mammalian nucleic acid 9 aaagcatggt ctctgtgtgt tctaatccctgttcattctc atttactgtc cctgggattt 60 cagatccaag accagcagga ctccagtcacctctacccca gctctccagg acacagcgct 120 cccaactctg agtgacgtcc cacctctggtccttgcagca caaccaacgt gggaatcaca 180 ccctccagac ctcccacagc tccaccccagactgggcgcc ggccctgcct ccatttcagc 240 tgtgacaacc tcagagccgt gttggcccaagcatgacaag gacgtatgaa aacttccagt 300 acttggagaa taaggtgaaa gtccaggggtttaaaaatgg gccacttcct ctccagtccc 360 tcctgctgct ggtcatcatc tgtgtggttggattccaaaa ttccaaattt cagagggacc 420 tggtgaccct gagaacagat tttagcaacttcacctcaaa cactgtggcg gagatccagg 480 cactgacttc ccagggcagc agcttggaagaaacgatagc atctctgaaa gctgaggtgg 540 agggtttcaa gcaggaacgg caggcagttcattctgaaat gctcctgcga gtccagcagc 600 tggtgcaaga cctgaagaaa ctgacctgccaggtggctac tctcaacaac aatggtgagg 660 aagcctccac tgaagggacc tgctgccccgtcaactgggt ggagcaccaa gacagctgct 720 actggttctc tcactctggg atgtcctgggccgaggctga gaagtactgc cagctgaaga 780 acgcccacct ggtggtcatc aactccagggaggagcagaa ttttgtccag aaatatctag 840 gctccgcata cacctggatg ggcctcagtgaccctgaagg agcctggaag tgggtggatg 900 gaacagacta tgcgaccggc ttccagaactggaagccagg ccagccagac gactggcagg 960 ggcacgggct gggtggaggc gaggactgtgctcacttcca tccagacggc aggtggaatg 1020 acgacgtctg ccagaggccc taccactgggtctgcgaggc tggcctgggt cagaccagcc 1080 aggagagtca ctgagctgcc tttggtgggaccacccggcc acagaaatgg cggtgggagg 1140 aggactcttc tcacgacctc ctcgcaagaccgctctggga gagaaataag cactgggaga 1200 ttggaagcac tgctaacatt ttgaatttttttctctttaa ttttaaaaag atggtatagt 1260 gttcttaagc ttttattttt tttccaacttttgaaagtca acttcatgaa ggtataattt 1320 ttacataata aaaatgcact catttaaagagtaaaaaaaa aaaaaaaaaa 1370 10 273 PRT Unknown mammalian protein 10 MetThr Arg Thr Tyr Glu Asn Phe Gln Tyr Leu Glu Asn Lys Val Lys 1 5 10 15Val Gln Gly Phe Lys Asn Gly Pro Leu Pro Leu Gln Ser Leu Leu Leu 20 25 30Leu Val Ile Ile Cys Val Val Gly Phe Gln Asn Ser Lys Phe Gln Arg 35 40 45Asp Leu Val Thr Leu Arg Thr Asp Phe Ser Asn Phe Thr Ser Asn Thr 50 55 60Val Ala Glu Ile Gln Ala Leu Thr Ser Gln Gly Ser Ser Leu Glu Glu 65 70 7580 Thr Ile Ala Ser Leu Lys Ala Glu Val Glu Gly Phe Lys Gln Glu Arg 85 9095 Gln Ala Val His Ser Glu Met Leu Leu Arg Val Gln Gln Leu Val Gln 100105 110 Asp Leu Lys Lys Leu Thr Cys Gln Val Ala Thr Leu Asn Asn Asn Gly115 120 125 Glu Glu Ala Ser Thr Glu Gly Thr Cys Cys Pro Val Asn Trp ValGlu 130 135 140 His Gln Asp Ser Cys Tyr Trp Phe Ser His Ser Gly Met SerTrp Ala 145 150 155 160 Glu Ala Glu Lys Tyr Cys Gln Leu Lys Asn Ala HisLeu Val Val Ile 165 170 175 Asn Ser Arg Glu Glu Gln Asn Phe Val Gln LysTyr Leu Gly Ser Ala 180 185 190 Tyr Thr Trp Met Gly Leu Ser Asp Pro GluGly Ala Trp Lys Trp Val 195 200 205 Asp Gly Thr Asp Tyr Ala Thr Gly PheGln Asn Trp Lys Pro Gly Gln 210 215 220 Pro Asp Asp Trp Gln Gly His GlyLeu Gly Gly Gly Glu Asp Cys Ala 225 230 235 240 His Phe His Pro Asp GlyArg Trp Asn Asp Asp Val Cys Gln Arg Pro 245 250 255 Tyr His Trp Val CysGlu Ala Gly Leu Gly Gln Thr Ser Gln Glu Ser 260 265 270 His 11 75 PRTUnknown mammalian protein 11 Glu Lys Met Ile Ile Lys Glu Leu Asn Tyr ThrGlu Leu Glu Cys Thr 1 5 10 15 Lys Trp Ala Ser Leu Leu Glu Asp Lys ValTrp Ser Cys Cys Pro Lys 20 25 30 Asp Trp Lys Pro Phe Gly Ser Tyr Cys TyrPhe Thr Ser Thr Asp Leu 35 40 45 Val Ala Ser Trp Asn Glu Ser Lys Glu AsnCys Phe His Met Gly Ala 50 55 60 His Leu Val Val Ile His Ser Gln Glu GluGln 65 70 75

What is claimed is:
 1. A substantially pure or recombinant DCMP1polypeptide exhibiting at least 85% sequence identity to SEQ ID NO: 2.2. A fusion protein comprising the polypeptide of claim
 1. 3. Arecombinant polypeptide comprising at least 10 contiguous amino acidresidues of residues 1-104 of SEQ ID NO:
 2. 4. A fusion proteincomprising the polypeptide of claim
 3. 5. A recombinant polypeptidecomprising the amino acid sequence of SEQ ID NO:
 2. 6. A fusion proteincomprising the polypeptide of claim
 5. 7. A recombinant DCMP1polypeptide consisting of the amino acid sequence of SEQ ID NO:
 2. 8. Afusion protein comprising the polypeptide of claim 7.